Convection-enhanced delivery of AAV vectors

ABSTRACT

Methods of delivering viral vectors, particularly recombinant AAV virions, to the CNS are provided. Also provided are methods of treating Parkinson&#39;s Disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is related to provisional patent applicationserial No. 60/086,949, filed May 27, 1998, and to provisional patentapplication, serial No. unassigned, entitled “Convection-EnhancedDelivery of AAV-AADC viral vector restores dopaminergic function inParkinsonian Monkeys,” filed May 18, 1999, from which applicationspriority is claimed under 35 USC §119(e)(1) and which are incorporatedherein by reference in their entireties.

FIELD OF THE INVENTION

[0002] The present invention relates generally to efficient delivery ofviral vectors to the CNS. More particularly, the present inventionrelates to gene therapy for the treatment of central nervous system(CNS) disorders, particularly those disorders which involve theneurotransmitter dopamine.

BACKGROUND OF THE INVENTION

[0003] CNS disorders are major public health issues. Parkinson's disease(PD) alone affects over 1 million people in the United States.Clinically, PD is characterized by a decrease in spontaneous movements,gait difficulty, postural instability, rigidity and tremor. Parkinson'sdisease is caused by the degeneration of the pigmented neurons in thesubstantia nigra of the brain, resulting in decreased dopamineavailability. Altered dopamine metabolism has also been implicated inschizophrenic patients who show increased dopamine in certain areas ofthe brain.

[0004] Currently, many CNS disorders such as PD are treated by systemicadministration of a therapeutic agent. Systemic administration, however,is often inefficient because of a drug's inability to pass through theblood brain barrier and because many drugs cause peripheral sideeffects. Thus, many potentially useful compounds, such as proteins,cannot be administered systemically. If these compounds are successfulin penetrating the blood-brain-barrier, they may also induce centralnervous system side effects as well. Treatment of PD currently involvesoral administration of the dopamine-precursor, L-dopa often incombination with a compound such ascarbidopa, a peripheral inhibitor ofthe enzyme aromatic amino acid decarboxylase (AADC) that decarboxylatesdopa to dopamine. In the majority of patients, however, production ofAADC in the affected brain regions is reduced as PD progresses and,consequently, larger and larger doses of L-dopa are required, leavingthe patients with reduced therapeutic benefits and increased sideeffects.

[0005] In view of the limitations of current systemic therapies, genedelivery is a promising method for the treatment for CNS disorders suchas PD. A number of viral based systems for gene transfer purposes havebeen described, such as retroviral systems which are currently the mostwidely used viral vector systems for this purpose. For descriptions ofvarious retroviral systems, see, e.g., U.S. Pat. No. 5,219,740; Millerand Rosman (1989) BioTechniques 7:980-990; Miller, A. D. (1990) HumanGene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns etal. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie andTemin (1993) Cur. Opin. Genet. Develop. 3:102-109.

[0006] Adeno-associated virus (AAV) systems are emerging as the leadingcandidates for use in gene therapy. AAV is a helper-dependent DNAparvovirus which belongs to the genus Dependovirus. AAV requiresinfection with an unrelated helper virus, either adenovirus, aherpesvirus or vaccinia, in order for a productive infection to occur.The helper virus supplies accessory functions that are necessary formost steps in AAV replication. For a review of AAV, see, e.g., Berns andBohenzky (1987) Advances in Virus Research (Academic Press, Inc.)32:243-307.

[0007] AAV infects a broad range of tissue, and has not elicited thecytotoxic effects and adverse immune reactions in animal models thathave been seen with other viral vectors. (see, e.g., Muzyczka, (1992)Current Topics in Microbiol. and Immunol. 158:97-129; Flotte et al.(1993) PNAS USA 90:10613-10617; Kass-eiser et al. (1992) Gene Therapy1:395-402; Yange et al. PNAS USA 91:4407-4411; Conrad et al. (1996) GeneTherapy 3:658-668; Yang et al. (1996) Gene Therapy 3:137-144; Brynes etal. (1996) J. Neurosci. 16:3045-3055). Because it can transducenondividing tissue, AAV may be well adapted for delivering genes to thecentral nervous system (CNS). U.S. Pat. Number 5,677,158 describedmethods of making AAV vectors. AAV vectors containing therapeutic genesunder the control of the cytomegalovirus (CMV) promoter have been shownto transduce mammalian brain and to have functional effects in models ofdisease.

[0008] AAV vectors carrying transgenes have been described, for example,in Kaplitt et al. (1994) Nature Genetics 8:148-153; WO 95/28493published Oct. 26, 1995; WO 95/34670, published Dec. 21, 1995; During etal., (1998) Gene Therapy 5:820-827; Mandel et al. (1998) J. Neurosci18:4271-4284; Szczypka et al. (1999) Neuron 22:167-178.). However,delivery of AAV vectors to the CNS has proven difficult. AAV has beenused to transfer the thymidine (tk) kinase gene to experimental gliomasin mice, and the ability of AAV-tk to render these brain tumorssensitive to the cytocidal effects of ganciclovir has been demonstrated.Okada et al. (1996) Gene Therapy 3:959-964; Mizuno et al. (1998) Jpn. JCancer Res. 89:76-80. Infusion of an AAV-CMV vector containing the humantyrosine hydroxylase (TH) gene, an enzyme involved in conversion of theamino acid tyrosine to dopa, into adult rat brain resulted intransduction of both neurons and glia (Kaplitt et al. (1995) VIRALVECTORS, GENE THERAPY AND NEUROSCIENCE APPLICATIONS, Kaplitt and Loewyeds., 12:193-210, Academic Press, San Diego; Bankiewicz et al. (1997)Exper. Neurol. 144:147-156). Delivery of the same vector to monkeystriatum resulted in robust expression of TH for up to 2.5 months(During et al., supra). Furthermore, AAV-CMV-TH was tested in a rodentmodel of Parkinson's Disease where it caused significant improvement inrotational behavior of 6-hydroxydopamine-lesioned rats (Fan et al.(1998) Human Gene Therapy 9:2527-2537; Mandel et al. (1997) PNAS USA94:14083-14088).

[0009] However, while reports such as these demonstrate AAV's potentialfor targeting the CNS, they also demonstrate that direct injection ofAAV vectors into the CNS results in limited numbers of transfected cellsand that the transfected cells are clustered in a narrow area near theinjection tracts. (see, e.g., During et al, supra; Fan et al., supra).Since multiple injections into the CNS cause undesirable complications,there remains a need for methods of delivering AAV vectors to largerareas of the brain using the least number of injection sites. Inaddition, the relationship between dose of injected vector and itsresulting distribution in brain tissue has not been previously reported.

[0010] Furthermore, gene therapy of PD has focused on delivery of atleast two genes encoding enzymes involved in dopamine synthesis, namelyTH and AADC. These methods are subject to all of the delivery problemsdiscussed above and, in addition, require that both genes are expressedin the proper amounts. Thus, treatment of PD using AAV-AADC incombination with L-dopa has also not been demonstrated.

SUMMARY OF THE INVENTION

[0011] In one aspect, the present invention provides methods fordelivering recombinant AAV (rAAV) virions carrying a transgene to thecentral nervous system (CNS) of a subject, for example a human, usingconvection-enhanced delivery (CED). CED can be conducted, for example,using either an osmotic pump or an infusion pump. In a preferredembodiment, the transgene encodes an aromatic amino acid decarboxylase(AADC) or active fragment thereof. When the transgene encodes an AADC,it is preferable to administer the rAAV virions into the striatum of theCNS.

[0012] In another aspect, the invention provides for methods fordelivering recombinant AAV virions to a subject having a CNS disorder.The rAAV virions encode a suitable therapeutic polypeptide and areadministered into the CNS of the subject using CED. In a preferredembodiment, the CNS disorder is Parkinson's disease (PD), the rAAVvirions are administered into the striatum of the CNS, and the nucleicacid sequence encodes AADC.

[0013] In another aspect, methods for treating a neurodegenerativedisease in a subject are provided. A preparation of recombinantadeno-associated virus (rAAV) virions carrying a therapeutic nucleicacid sequence that is expressible in transduced cells is administered tothe CNS using convection-enhanced delivery (CED). In one embodiment, theneurodegenerative disease is PD and the therapeutic polypeptide is anAADC. In yet another embodiment, the method of treating theneurodegenative disease also includes administering at least oneadditional therapeutic compound to the subject, for example,systemically administering L-dopa and, optionally, carbidopa.

[0014] In yet another aspect, methods of determining levels of dopamineactivity in the CNS of subject are provided. A labeled tracer isadministered to the subject. The tracer is preferably a compound thatbinds to a cell which utilizes dopamine and the label is preferably aradioisotope, for instance, 6-[¹⁸F]-fluoro-L-m-tyrosine (¹⁸F-FMT). Thedetection of the label is indicative of dopamine activity via binding ofthe tracer. Preferably, the subject's CNS is imaged, for example usingpositron emission tomography (PET) scanning.

[0015] These and other embodiments of the subject invention will readilyoccur to those of ordinary skill in the art in view of the disclosureherein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]FIG. 1, panels a though d, depict dose responses (expression ofthe AAV-tk transgene) in rat following intracranial infusion pumpdelivery. The tissue volume (FIG. 1a); mean area (FIG. 1b); length (FIG.1c) and number of cells (FIG. 1d) expressing the transgene are depicted.

[0017]FIG. 2 is a half-tone reproduction showing labeling of rat braintissue after injection of AAV vectors.

[0018]FIG. 3, panels a though d, depict intracranial delivery of AAV-tkthrough either an infusion pump (IP) or osmotic pump (OP). The tissuevolume (FIG. 3a); mean area (FIG. 3b); length (FIG. 3c) and number ofcells (FIG. 3d) expressing the transgene are depicted.

[0019]FIGS. 4a, 4 b, 4 c and 4 d are half-tone reproductions depictingCNS tissue infused with with vector carrying the tk transgene. FIGS. 4aand b show expression of tk in neurons. FIGS. 4c and d show expressionin neurons and glial near the site of osmotic pump infusion.

[0020]FIG. 5 is a half-tone reproduction depicting Southern blotanalysis of tissues from a subject infused with AAV-tk vector.

[0021]FIG. 6 depicts immunostaining for AADC of the brains ofMPTP-lesioned monkeys. The left side (control) shows limited staining,while the right side (AAV-AADC treated) shows broad AADC immunostaining.

[0022]FIG. 7 is an FMT PET scan depicting dopamine activity in thebrains of unilaterally MPTP-lesioned monkeys. The left side (baseline)shows limited activity on the lesioned side, while the right side (8weeks post AAV-AADC administration) shows normal levels of dopamineactivity.

[0023]FIG. 8, panels A though C depict biochemical analysis of L-dopalevels in MPTP-lesioned monkeys. Panel A shows that L-dopa is convertedto dopamine by the AACD enzyme. In cortical regions, regardless of theMPTP treatment, there is poor or no conversion of L-dopa to dopamine.Striatum is AADC-rich, therefore, most of the L-dopa has been convertedto dopamine in this region. On the striatum ipsilateral to MPTPadministration, L-dopa conversion to dopamine is impaired and similar tocortical activity in AAV-LacZ treated monkeys. Both AAV-AADC-treatedanimals show almost normal rates of L-dopa to dopamine conversion. PanelB depicts HVA analysis. HVA is a metabolite of dopamine catabolism.Since cortical regions are not able to convert L-dopa to dopamine HVAlevels are low. As shown in panel A, striatum converts L-dopa todopamine, therefore, dopamine is catabolised to HVA in this region.Since AADC activity has not been restored in the AAV-LacZ-treatedmonkeys HVA levels in MTPT ipsilateral striatum are low. HVA levels aresignificantly elevated in AAV-AADC-treated monkey in the MPTPipsilateral striatum. Panel C shows L-dopa levels were measured in thetissue punches following L-dopa administration. Due to the differentL-dopa absorption tissue levels differ between the monkeys. They aresimilar, however, within each subject. Tissue levels of L-dopa weredramatically reduced in the MPTP ipsilateral stratum of AAV-AADC-treatedmonkeys, since AADC enzyme has been restored. The activity of AADC inthis region is very strong, since tissue levels of L-dopa are lower thanin the contralateral striatum.

[0024]FIG. 9 is a graph depicting activity of the AADC enzyme in-vitro.Tissue punches were incubated with L-dopa as described in the materialand methods. AADC enzyme activity was determined by measuring rates ofL-dopa to dopamine conversion. Cortical regions contain low levels ofAADC. AADC activity in contralateral striatum is high, however it isvariable since there is some dopaminergic lesion on that side of thebrain. AADC activity in MPTP ipsilatral striatum is significantlyreduced in AAV-Lac-Z-treated monkey while it is completely restored inthe AAV-DDC monkeys.

DETAILED DESCRIPTION OF THE INVENTION

[0025] The practice of the present invention will employ, unlessotherwise indicated, conventional methods of virology, microbiology,molecular biology and recombinant DNA techniques within the skill of theart. Such techniques are explained fully in the literature. See, e.g.,Sambrook, et al. Molecular Cloning: A Laboratory Manual (CurrentEdition); Current Protocols in Molecular Biology (F. M. Ausubel, et al.eds., current edition); DNA Cloning: A Practical Approach, vol. I & II(D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., CurrentEdition); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds.,Current Edition); Transcription and Translation (B. Hames & S. Higgins,eds., Current Edition); CRC Handbook of Parvoviruses, vol. I & II (P.Tijessen, ed.); Fundamental Virology, 2nd Edition, vol. I & II (B. N.Fields and D. M. Knipe, eds.)

[0026] All publications, patents and patent applications cited herein,whether supra or infra, are hereby incorporated by reference in theirentirety.

[0027] As used in this specification and the appended claims, thesingular forms “a,” “an” and “the” include plural references unless thecontent clearly dictates otherwise.

Definitions

[0028] In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

[0029] “Gene transfer” or “gene delivery” refers to methods or systemsfor reliably inserting foreign DNA into host cells. Such methods canresult in transient expression of non-integrated transferred DNA,extrachromosomal replication and expression of transferred replicons(e.g., episomes), or integration of transferred genetic material intothe genomic DNA of host cells. Gene transfer provides a unique approachfor the treatment of acquired and inherited diseases. A number ofsystems have been developed for gene transfer into mammalian cells. See,e.g., U.S. Pat. No. 5,399,346.

[0030] By “vector” is meant any genetic element, such as a plasmid,phage, transposon, cosmid, chromosome, virus, virion, etc., which iscapable of replication when associated with the proper control elementsand which can transfer gene sequences between cells. Thus, the termincludes cloning and expression vehicles, as well as viral vectors.

[0031] By “recombinant virus” is meant a virus that has been geneticallyaltered, e.g., by the addition or insertion of a heterologous nucleicacid construct into the particle.

[0032] By “AAV virion” is meant a complete virus particle, such as awild-type (wt) AAV virus particle (comprising a linear, single-strandedAAV nucleic acid genome associated with an AAV capsid protein coat). Inthis regard, single-stranded AAV nucleic acid molecules of eithercomplementary sense, e.g., “sense” or “antisense” strands, can bepackaged into any one AAV virion and both strands are equallyinfectious.

[0033] A “recombinant AAV virion,” or “rAAV virion” is defined herein asan infectious, replication-defective virus composed of an AAV proteinshell, encapsidating a heterologous nucleotide sequence of interestwhich is flanked on both sides by AAV ITRs. A rAAV virion is produced ina suitable host cell which has had an AAV vector, AAV helper functionsand accessory functions introduced therein. In this manner, the hostcell is rendered capable of encoding AAV polypeptides that are requiredfor packaging the AAV vector (containing a recombinant nucleotidesequence of interest) into infectious recombinant virion particles forsubsequent gene delivery.

[0034] The term “transfection” is used to refer to the uptake of foreignDNA by a cell, and a cell has been “transfected” when exogenous DNA hasbeen introduced inside the cell membrane. A number of transfectiontechniques are generally known in the art. See, e.g., Graham et al.(1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, alaboratory manual, Cold Spring Harbor Laboratories, New York, Davis etal. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al.(1981) Gene 13:197. Such techniques can be used to introduce one or moreexogenous DNA moieties, such as a nucleotide integration vector andother nucleic acid molecules, into suitable host cells.

[0035] The term “host cell” denotes, for example, microorganisms, yeastcells, insect cells, and mammalian cells, that can be, or have been,used as recipients of an AAV helper construct, an AAV vector plasmid, anaccessory function vector, or other transfer DNA. The term includes theprogeny of the original cell which has been transfected. Thus, a “hostcell” as used herein generally refers to a cell which has beentransfected with an exogenous DNA sequence. It is understood that theprogeny of a single parental cell may not necessarily be completelyidentical in morphology or in genomic or total DNA complement as theoriginal parent, due to natural, accidental, or deliberate mutation.

[0036] As used herein, the term “cell line” refers to a population ofcells capable of continuous or prolonged growth and division in vitro.Often, cell lines are clonal populations derived from a singleprogenitor cell. It is further known in the art that spontaneous orinduced changes can occur in karyotype during storage or transfer ofsuch clonal populations. Therefore, cells derived from the cell linereferred to may not be precisely identical to the ancestral cells orcultures, and the cell line referred to includes such variants.

[0037] The term “heterologous” as it relates to nucleic acid sequencessuch as coding sequences and control sequences, denotes sequences thatare not normally joined together, and/or are not normally associatedwith a particular cell. Thus, a “heterologous” region of a nucleic acidconstruct or a vector is a segment of nucleic acid within or attached toanother nucleic acid molecule that is not found in association with theother molecule in nature. For example, a heterologous region of anucleic acid construct could include a coding sequence flanked bysequences not found in association with the coding sequence in nature.Another example of a heterologous coding sequence is a construct wherethe coding sequence itself is not found in nature (e.g., syntheticsequences having codons different from the native gene). Similarly, acell transformed with a construct which is not normally present in thecell would be considered heterologous for purposes of this invention.Allelic variation or naturally occurring mutational events do not giverise to heterologous DNA, as used herein.

[0038] A “coding sequence” or a sequence which “encodes” a particularprotein, is a nucleic acid sequence which is transcribed (in the case ofDNA) and translated (in the case of mRNA) into a polypeptide in vitro orin vivo when placed under the control of appropriate regulatorysequences. The boundaries of the coding sequence are determined by astart codon at the 5′ (amino) terminus and a translation stop codon atthe 3′ (carboxy) terminus. A coding sequence can include, but is notlimited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNAsequences from prokaryotic or eukaryotic DNA, and even synthetic DNAsequences. A transcription termination sequence will usually be located3′ to the coding sequence.

[0039] A “nucleic acid” sequence refers to a DNA or RNA sequence. Theterm captures sequences that include any of the known base analogues ofDNA and RNA such as, but not limited to 4-acetylcytosine,8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine,5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethylaminomethyluracil, dihydrouracil, inosine,N6-isopentenyladenine, 1 -methyladenine, 1 -methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and2,6-diaminopurine.

[0040] The term DNA “control sequences” refers collectively to promotersequences, polyadenylation signals, transcription termination sequences,upstream regulatory domains, origins of replication, internal ribosomeentry sites (“IRES”), enhancers, and the like, which collectivelyprovide for the replication, transcription and translation of a codingsequence in a recipient cell. Not all of these control sequences needalways be present so long as the selected coding sequence is capable ofbeing replicated, transcribed and translated in an appropriate hostcell.

[0041] The term “promoter region” is used herein in its ordinary senseto refer to a nucleotide region comprising a DNA regulatory sequence,wherein the regulatory sequence is derived from a gene which is capableof binding RNA polymerase and initiating transcription of a downstream(3′-direction) coding sequence.

[0042] “Operably linked” refers to an arrangement of elements whereinthe components so described are configured so as to perform their usualfunction. Thus, control sequences operably linked to a coding sequenceare capable of effecting the expression of the coding sequence. Thecontrol sequences need not be contiguous with the coding sequence, solong as they function to direct the expression thereof. Thus, forexample, intervening untranslated yet transcribed sequences can bepresent between a promoter sequence and the coding sequence and thepromoter sequence can still be considered “operably linked” to thecoding sequence.

[0043] By “isolated” when referring to a nucleotide sequence, is meantthat the indicated molecule is present in the substantial absence ofother biological macromolecules of the same type. Thus, an “isolatednucleic acid molecule which encodes a particular polypeptide” refers toa nucleic acid molecule which is substantially free of other nucleicacid molecules that do not encode the subject polypeptide; however, themolecule may include some additional bases or moieties which do notdeleteriously affect the basic characteristics of the composition.

[0044] For the purpose of describing the relative position of nucleotidesequences in a particular nucleic acid molecule throughout the instantapplication, such as when a particular nucleotide sequence is describedas being situated “upstream,” “downstream,” “3′,” or “5″” relative toanother sequence, it is to be understood that it is the position of thesequences in the “sense” or “coding” strand of a DNA molecule that isbeing referred to as is conventional in the art.

[0045] A “gene” refers to a polynucleotide containing at least one openreading frame that is capable of encoding a particular polypeptide orprotein after being transcribed or translated. Any of the polynucleotidesequences described herein may be used to identify larger fragments orfull-length coding sequences of the genes with which they areassociated. Methods of isolating larger fragment sequences are know tothose of skill in the art.

[0046] Two nucleic acid fragments are considered to “selectivelyhybridize” as described herein. The degree of sequence identity betweentwo nucleic acid molecules affects the efficiency and strength ofhybridization events between such molecules. A partially identicalnucleic acid sequence will at least partially inhibit a completelyidentical sequence from hybridizing to a target molecule. Inhibition ofhybridization of the completely identical sequence can be assessed usinghybridization assays that are well known in the art (e.g., Southernblot, Northern blot, solution hybridization, or the like, see Sambrook,et al., Molecular Cloning: A Laboratory Manual, Second Edition, (1989)Cold Spring Harbor, N.Y.). Such assays can be conducted using varyingdegrees of selectivity, for example, using conditions varying from lowto high stringency. If conditions of low stringency are employed, theabsence of non-specific binding can be assessed using a secondary probethat lacks even a partial degree of sequence identity (for example, aprobe having less than about 30% sequence identity with the targetmolecule), such that, in the absence of non-specific binding events, thesecondary probe will not hybridize to the target.

[0047] When utilizing a hybridization-based detection system, a nucleicacid probe is chosen that is complementary to a target nucleic acidsequence, and then by selection of appropriate conditions the probe andthe target sequence “selectively hybridize,” or bind, to each other toform a hybrid molecule. A nucleic acid molecule that is capable ofhybridizing selectively to a target sequence under “moderatelystringent” conditions typically hybridizes under conditions that allowdetection of a target nucleic acid sequence of at least about 10-14nucleotides in length having at least approximately 70% sequenceidentity with the sequence of the selected nucleic acid probe. Stringenthybridization conditions typically allow detection of target nucleicacid sequences of at least about 10-14 nucleotides in length having asequence identity of greater than about 90-95% with the sequence of theselected nucleic acid probe. Hybridization conditions useful forprobe/target hybridization where the probe and target have a specificdegree of sequence identity, can be determined as is known in the art(see, for example, Nucleic Acid Hybridization: A Practical Approacheditors B. D. Hames and S. J. Higgins, (1985) Oxford; Washington, DC;IRL Press).

[0048] With respect to stringency conditions for hybridization, it iswell known in the art that numerous equivalent conditions can beemployed to establish a particular stringency by varying, for example,the following factors: the length and nature of probe and targetsequences, base composition of the various sequences, concentrations ofsalts and other hybridization solution components, the presence orabsence of blocking agents in the hybridization solutions (e.g.,formamide, dextran sulfate, and polyethylene glycol), hybridizationreaction temperature and time parameters, as well as, varying washconditions. The selection of a particular set of hybridizationconditions is selected following standard methods in the art (see, forexample, Sambrook, et al., Molecular Cloning: A Laboratory Manual,Second Edition, (1989) Cold Spring Harbor, N.Y.).

[0049] The term “aromatic amino acid decarboxylase” or “AADC” refers toa polypeptide which decarboxylates dopa to dopamine. Thus, the termincludes a full-length AADC polypeptide, active fragments or functionalhomologues thereof.

[0050] A “functional homologue,” or a “functional equivalent” of a givenpolypeptide includes molecules derived from the native polypeptidesequence, as well as recombinantly produced or chemically synthesizedpolypeptides which function in a manner similar to the referencemolecule to achieve a desired result. Thus, a functional homologue ofAADC encompasses derivatives and analogues of thosepolypeptides—including any single or multiple amino acid additions,substitutions and/or deletions occurring internally or at the amino orcarboxy termini thereof—so long as integrity of activity remains.

[0051] Techniques for determining nucleic acid and amino acid “sequenceidentity” or “homology” also are known in the art. Typically, suchtechniques include determining the nucleotide sequence of the mRNA for agene and/or determining the amino acid sequence encoded thereby, andcomparing these sequences to a second nucleotide or amino acid sequence.In general, “identity” refers to an exact nucleotide-to-nucleotide oramino acid-to-amino acid correspondence of two polynucleotides orpolypeptide sequences, respectively. Two or more sequences(polynucleotide or amino acid) can be compared by determining their“percent identity.” The percent identity of two sequences, whethernucleic acid or amino acid sequences, is the number of exact matchesbetween two aligned sequences divided by the length of the shortersequences and multiplied by 100. An approximate alignment for nucleicacid sequences is provided by the local homology algorithm of Smith andWaterman, Advances in Applied Mathematics 2:482-489 (1981). Thisalgorithm can be applied to amino acid sequences by using the scoringmatrix developed by Dayhoff, Atlas of Protein Seguences and Structure,M. O. Dayhoff ed., 5 suppl. 3:353-358, National Biomedical ResearchFoundation, Washington, D.C., USA, and normalized by Gribskov, Nucl.Acids Res. 14(6):6745-6763 (1986). An exemplary implementation of thisalgorithm to determine percent identity of a sequence is provided by theGenetics Computer Group (Madison, Wis.) in the “BestFit” utilityapplication. The default parameters for this method are described in theWisconsin Sequence Analysis Package Program Manual, Version 8 (1995)(available from Genetics Computer Group, Madison, Wis.). A preferredmethod of establishing percent identity in the context of the presentinvention is to use the MPSRCH package of programs copyrighted by theUniversity of Edinburgh, developed by John F. Collins and Shane S.Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View,Calif.). From this suite of packages the Smith-Waterman algorithm can beemployed where default parameters are used for the scoring table (forexample, gap open penalty of 12, gap extension penalty of one, and a gapof six). From the data generated the “Match” value reflects “sequenceidentity.” Other suitable programs for calculating the percent identityor similarity between sequences are generally known in the art, forexample, another alignment program is BLAST, used with defaultparameters. For example, BLASTN and BLASTP can be used using thefollowing default parameters: genetic code=standard; filter=none;strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50sequences; sort by=HIGH SCORE; Databases=non-redundant,GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swissprotein+Spupdate+PIR. Details of these programs can be found at thefollowing internet address: http://www.ncbi.nlm.gov/cgi-bin/BLAST.

[0052] Alternatively, homology can be determined by hybridization ofpolynucleotides under conditions which form stable duplexes betweenhomologous regions, followed by digestion with single-stranded-specificnuclease(s), and size determination of the digested fragments. Two DNA,or two polypeptide sequences are “substantially homologous” to eachother when the sequences exhibit at least about 80%-85%, preferably atleast about 90%, and most preferably at least about 95%-98% sequenceidentity over a defined length of the molecules, as determined using themethods above. As used herein, substantially homologous also refers tosequences showing complete identity to the specified DNA or polypeptidesequence. DNA sequences that are substantially homologous can beidentified in a Southern hybridization experiment under, for example,stringent conditions, as defined for that particular system. Definingappropriate hybridization conditions is within the skill of the art.See, e.g., Sambrook et al., supra; DNA Cloning, supra; Nucleic AcidHybridization, supra.

[0053] “Convection-enhanced delivery” refers to any non-manual deliveryof agents. In the context of the present invention, examples ofconvection-enhanced delivery (CED) of AAV can be achieved by infusionpumps or by osmotic pumps.

[0054] The term “central nervous system” or “CNS” includes all cells andtissue of the brain and spinal cord of a vertebrate. Thus, the termincludes, but is not limited to, neuronal cells, glial cells,astrocytes, cereobrospinal fluid (CSF), interstitial spaces, bone,cartilage and the like. The “cranial cavity” refers to the areaunderneath the skull (cranium). Regions of the CNS have been associatedwith various behaviors and/or functions. For example, the basal gangliaof the brain has been associated with motor functions, particularlyvoluntary movement. The basal ganglia is composed of six paired nuclei:the caudate nucleus, the putamen, the globus pallidus (or pallidum), thenucleus accumbens, the subthalamic nucleus and the substantia nigra. Thecaudate nucleus and putamen, although separated by the internal capsula,share cytoarchitechtonic, chemical and physiologic properties and areoften referred to as the corpus striatum, or simply “the striatum.” Thesubstantia nigra, which degenerates in Parkinson's patients, providesmajor dopaminergic input into the basal ganglia.

[0055] The terms “subject”, “individual” or “patient” are usedinterchangeably herein and refer to a vertebrate, preferably a mammal.Mammals include, but are not limited to, murines, simians, humans, farmanimals, sport animals and pets.

[0056] An “effective amount” is an amount sufficient to effectbeneficial or desired results. An effective amount can be administeredin one or more administrations, applications or dosages.

[0057] The term “labeled tracer” refers to any molecule which can beused to follow or detect a defined activity in vivo, for example, apreferred tracer is one that binds to cells that are utilizing dopamine.Preferably, the labeled tracer is one that can be viewed in a wholeanimal, for example, by positron emission tomograph (PET) scanning orother CNS imaging techniques. Suitable labels include, but are notlimited to radioisotopes, fluorochromes, chemiluminescent compounds,dyes, and proteins, including enzymes.

GENERAL OVERVIEW OF THE INVENTION

[0058] Central to the present invention is the development of methodswhich allow for the efficient delivery of viral vectors, such as AAV,into the CNS of animal. Previously, researchers have had only minimalsuccess delivering viral vectors to widespread areas of the brain. Usingconvection-enhanced delivery devices (for example, osmotic or infusionpumps), viral vectors can be delivered to many cells over large areas ofthe brain. Moreover, the delivered vectors efficiently expresstransgenes in CNS cells (e.g., neurons or glial cells).

[0059] Using the methods of viral vector delivery described herein,novel gene therapy treatments for CNS disorders (e.g., Parkinson'sDisease) can be devised. In one embodiment, Parkinson's disease (PD) istreated by combining systemic L-dopa and/or carbidopa therapy withCNS-administration (e.g., via CED) of AAV vectors carrying a transgeneencoding AADC, an enzyme involved in dopamine metabolism.

[0060] Advantages of the invention, include, but are not limited to (i)efficient and widespread delivery of viral vectors (such as AAV) to theCNS; (ii) expression of nucleic acids (e.g., transgenes) carried by theviral vectors; (iii) identification of a therapeutic regime forParkinson's Disease that involves delivery of one transgene incombination with administration of a pro-drug; and (iv) the ability tonon-invasively monitor CNS gene therapy using PET scan.

Construction of Viral Vectors

[0061] Gene delivery vehicles useful in the practice of the presentinvention can be constructed utilizing methodologies well known in theart of molecular biology (see, for example, Ausubel or Maniatis, supra).Typically, viral vectors carrying transgenes are assembled frompolynuclotides encoding the transgene(s), suitable regulatory elementsand elements necessary for production of viral proteins which mediatecell transduction. For example, in a preferred embodiment,adeno-associated viral (AAV) vectors are employed.

[0062] General Methods

[0063] A preferred method of obtaining the nucleotide components of theviral vector is PCR. General procedures for PCR are taught in MacPhersonet al., PCR: A PRACTICAL APPROACH, (IRL Press at Oxford UniversityPress, (1991)). PCR conditions for each application reaction may beempirically determined. A number of parameters influence the success ofa reaction. Among these parameters are annealing temperature and time,extension time, Mg²⁺and ATP concentration, pH, and the relativeconcentration of primers, templates and deoxyribonucleotides. Exemplaryprimers are described below in the Examples. After amplification, theresulting fragments can be detected by agarose gel electrophoresisfollowed by visualization with ethidium bromide staining and ultravioletillumination.

[0064] Another method for obtaining polynucleotides is by enzymaticdigestion. For example, nucleotide sequences can be generated bydigestion of appropriate vectors with suitable recognition restrictionenzymes. The resulting fragments can then be ligated together asappropriate.

[0065] Polynucleotides are inserted into vector genomes using methodswell known in the art. For example, insert and vector DNA can becontacted, under suitable conditions, with a restriction enzyme tocreate complementary or blunt ends on each molecule that can pair witheach other and be joined with a ligase. Alternatively, synthetic nucleicacid linkers can be ligated to the termini of a polynucleotide. Thesesynthetic linkers can contain nucleic acid sequences that correspond toa particular restriction site in the vector DNA. Other means are knownand available in the art.

[0066] Retroviral and Adenoviral Vectors

[0067] A number of viral based systems have been used for gene delivery.For example, retroviral systems are known and generally employ packaginglines which have an integrated defective provirus (the “helper” ) thatexpresses all of the genes of the virus but cannot package its owngenome due to a deletion of the packaging signal, known as the psisequence. Thus, the cell line produces empty viral shells. Producerlines can be derived from the packaging lines which, in addition to thehelper, contain a viral vector which includes sequences required in cisfor replication and packaging of the virus, known as the long terminalrepeats (LTRs). The gene of interest can be inserted in the vector andpackaged in the viral shells synthesized by the retroviral helper. Therecombinant virus can then be isolated and delivered to a subject. (See,e.g., U.S. Pat. No. 5,219,740.) Representative retroviral vectorsinclude but are not limited to vectors such as the LHL, N2, LNSAL, LSHLand LHL2 vectors described in e.g., U.S. Pat. No. 5,219,740,incorporated herein by reference in its entirety, as well as derivativesof these vectors, such as the modified N2 vector described herein.Retroviral vectors can be constructed using techniques well known in theart. See, e.g., U.S. Pat. No 5,219,740; Mann et al. (1983) Cell33:153-159.

[0068] Adenovirus based systems have been developed for gene deliveryand are suitable for delivery according to the methods described herein.Human adenoviruses are double-stranded DNA viruses which enter cells byreceptor-mediated endocytosis. These viruses are particularly wellsuited for gene transfer because they are easy to grow and manipulateand they exhibit a broad host range in vivo and in vitro. For example,adenoviruses can infect human cells of hematopoietic, lymphoid andmyeloid origin. Furthermore, adenoviruses infect quiescent as well asreplicating target cells. Unlike retroviruses which integrate into thehost genome, adenoviruses persist extrachromosomally thus minimizing therisks associated with insertional mutagenesis. The virus is easilyproduced at high titers and is stable so that it can be purified andstored. Even in the replication-competent form, adenoviruses cause onlylow level morbidity and are not associated with human malignancies.Accordingly, adenovirus vectors have been developed which make use ofthese advantages. For a description of adenovirus vectors and their usessee, e.g., Haj-Ahmad and Graham (1986) J. Virol. 57:267-274; Bett et al.(1993) J. Virol. 67:5911-5921; Mittereder et al. (1994) Human GeneTherapy 5:717-729; Seth et al. (1994) J. Virol. 68:933-940; Barr et al.(1994) Gene Therapy 1:51-58; Berkner, K. L. (1988) BioTechniques6:616-629;Rich et al. (1993) Human Gene Therapy 4:461476.

[0069] AAV Expression Vectors

[0070] In a preferred embodiment, the viral vectors are AAV vectors. Byan “AAV vector” is meant a vector derived from an adeno-associated virusserotype, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4,AAV-5, AAVX7, etc. AAV vectors can have one or more of the AAV wild-typegenes deleted in whole or part, preferably the rep and/or cap genes, butretain functional flanking ITR sequences. Functional ITR sequences arenecessary for the rescue, replication and packaging of the AAV virion.Thus, an AAV vector is defined herein to include at least thosesequences required in cis for replication and packaging (e.g.,functional ITRs) of the virus. The ITRs need not be the wild-typenucleotide sequences, and may be altered, e.g., by the insertion,deletion or substitution of nucleotides, so long as the sequencesprovide for functional rescue, replication and packaging.

[0071] AAV expression vectors are constructed using known techniques toat least provide as operatively linked components in the direction oftranscription, control elements including a transcriptional initiationregion, the DNA of interest and a transcriptional termination region.The control elements are selected to be functional in a mammalian cell.The resulting construct which contains the operatively linked componentsis bounded (5′ and 3′) with functional AAV ITR sequences.

[0072] By “adeno-associated virus inverted terminal repeats” or “AAVITRs” is meant the art-recognized regions found at each end of the AAVgenome which function together in cis as origins of DNA replication andas packaging signals for the virus. AAV ITRs, together with the AAV repcoding region, provide for the efficient excision and rescue from, andintegration of a nucleotide sequence interposed between two flankingITRs into a mammalian cell genome.

[0073] The nucleotide sequences of AAV ITR regions are known. See, e.g.,Kotin, R. M. (1994) Human Gene Therapy 5:793-801; Berns, K. I.“Parvoviridae and their Replication” in Fundamental Virology, 2ndEdition, (B. N. Fields and D. M. Knipe, eds.) for the AAV-2 sequence. Asused herein, an “AAV ITR” need not have the wild-type nucleotidesequence depicted, but may be altered, e.g., by the insertion, deletionor substitution of nucleotides. Additionally, the AAV ITR may be derivedfrom any of several AAV serotypes, including without limitation, AAV-1,AAV-2, AAV-3, AAV-4, AAV-5, AAVX7, etc. Furthermore, 5′ and 3′ ITRswhich flank a selected nucleotide sequence in an AAV vector need notnecessarily be identical or derived from the same AAV serotype orisolate, so long as they function as intended, i.e., to allow forexcision and rescue of the sequence of interest from a host cell genomeor vector, and to allow integration of the heterologous sequence intothe recipient cell genome when AAV Rep gene products are present in thecell.

[0074] Additionally, AAV ITRs may be derived from any of several AAVserotypes, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4,AAV-5, AAVX7, etc. Furthermore, 5′ and 3′ ITRs which flank a selectednucleotide sequence in an AAV expression vector need not necessarily beidentical or derived from the same AAV serotype or isolate, so long asthey function as intended, i.e., to allow for excision and rescue of thesequence of interest from a host cell genome or vector, and to allowintegration of the DNA molecule into the recipient cell genome when AAVRep gene products are present in the cell.

[0075] Suitable DNA molecules for use in AAV vectors will be less thanabout 5 kilobases (kb) in size and will include, for example, a genethat encodes a protein that is defective or missing from a recipientsubject or a gene that encodes a protein having a desired biological ortherapeutic effect (e.g., an antibacterial, antiviral or antitumorfunction). Preferred DNA molecules include those involved in dopaminemetabolism, for example, AADC or TH. AAV-AADC and AAV-TH vectors havebeen described, for example, in Bankiewicz et al. (1997) Exper't Neurol.144:147-156; Fan et al (1998) Human Gene Therapy 9:2527-2535 andInternational Publication WO 95/28493, published Oct. 26, 1995.

[0076] The selected nucleotide sequence, such as AADC or another gene ofinterest, is operably linked to control elements that direct thetranscription or expression thereof in the subject in vivo. Such controlelements can comprise control sequences normally associated with theselected gene. Alternatively, heterologous control sequences can beemployed. Useful heterologous control sequences generally include thosederived from sequences encoding mammalian or viral genes. Examplesinclude, but are not limited to, the SV40 early promoter, mouse mammarytumor virus LTR promoter; adenovirus major late promoter (Ad MLP); aherpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promotersuch as the CMV immediate early promoter region (CMVIE), a rous sarcomavirus (RSV) promoter, synthetic promoters, hybrid promoters, and thelike. In addition, sequences derived from nonviral genes, such as themurine metallothionein gene, will also find use herein. Such promotersequences are commercially available from, e.g., Stratagene (San Diego,Calif.).

[0077] For purposes of the present invention, both heterologouspromoters and other control elements, such as CNS-specific and induciblepromoters, enhancers and the like, will be of particular use. Examplesof heterologous promoters include the CMB promoter. Examples ofCNS-specific promoters include those isolated from the genes from myelinbasic protein (MBP), glial fibrillary acid protein (GFAP), and neuronspecific enolase (NSE). Examples of inducible promoters include DNAresponsive elements for ecdysone, tetracycline, hypoxia and aufin.

[0078] The AAV expression vector which harbors the DNA molecule ofinterest bounded by AAV ITRs, can be constructed by directly insertingthe selected sequence(s) into an AAV genome which has had the major AAVopen reading frames (“ORFs”) excised therefrom. Other portions of theAAV genome can also be deleted, so long as a sufficient portion of theITRs remain to allow for replication and packaging functions. Suchconstructs can be designed using techniques well known in the art. See,e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941; International PublicationNos. WO 92/01070 (published Jan. 23, 1992) and WO 93/03769 (publishedMar. 4 1993); Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996;Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press);Carter, B. J. (1992) Current Opinion in Biotechnology 3:533-539;Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol.158:97-129; Kotin, R. M. (1994) Human Gene Therapy 5:793-801; Shellingand Smith (1994) Gene Therapy 1:165-169; and Zhou et al. (1994) J. Exp.Med. 179:1867-1875.

[0079] Alternatively, AAV ITRs can be excised from the viral genome orfrom an AAV vector containing the same and fused 5′ and 3′ of a selectednucleic acid construct that is present in another vector using standardligation techniques, such as those described in Sambrook et al., supra.For example, ligations can be accomplished in 20 mM Tris-Cl pH 7.5, 10mM MgCl₂, 10 mM DTT, 33 ug/ml BSA, 10 mM-50 mM NaCl, and either 40 uMATP, 0.01-0.02 (Weiss) units T4 DNA ligase at 0° C. (for “sticky end”ligation) or 1 mM ATP, 0.3-0.6 (Weiss) units T4 DNA ligase at 14° C.(for “blunt end” ligation). Intermolecular “sticky end” ligations areusually performed at 30-100 μg/ml total DNA concentrations (5-100 nMtotal end concentration). AAV vectors which contain ITRs have beendescribed in, e.g., U.S. Pat. No. 5,139,941. In particular, several AAVvectors are described therein which are available from the American TypeCulture Collection (“ATCC” ) under Accession Numbers 53222, 53223,53224, 53225 and 53226.

[0080] Additionally, chimeric genes can be produced synthetically toinclude AAV ITR sequences arranged 5′ and 3′ of one or more selectednucleic acid sequences. Preferred codons for expression of the chimericgene sequence in mammalian CNS cells can be used. The complete chimericsequence is assembled from overlapping oligonucleotides prepared bystandard methods. See, e.g., Edge, Nature (1981) 292:756; Nambair et al.Science (1984) 223:1299; Jay et al. J. Biol. Chem. (1984) 259:6311.

[0081] In order to produce rAAV virions, an AAV expression vector isintroduced into a suitable host cell using known techniques, such as bytransfection. A number of transfection techniques are generally known inthe art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook etal. (1989) Molecular Cloning, a laboratory manual, Cold Spring HarborLaboratories, New York, Davis et al. (1986) Basic Methods in MolecularBiology, Elsevier, and Chu et al. (1981) Gene 13:197. Particularlysuitable transfection methods include calcium phosphate co-precipitation(Graham et al. (1973) Virol. 52:456-467), direct micro-injection intocultured cells (Capecchi, M. R. (1980) Cell 22:479-488), electroporation(Shigekawa et al. (1988) BioTechniques 6:742-751), liposome mediatedgene transfer (Mannino et al. (1988) BioTechniques 6:682-690),lipid-mediated transduction (Felgner et al. (1987) Proc. Natl. Acad.Sci. USA 84:7413-7417), and nucleic acid delivery using high-velocitymicroprojectiles (Klein et al. (1987) Nature 327:70-73).

[0082] For the purposes of the invention, suitable host cells forproducing rAAV virions include microorganisms, yeast cells, insectcells, and mammalian cells, that can be, or have been, used asrecipients of a heterologous DNA molecule. The term includes the progenyof the original cell which has been transfected. Thus, a “host cell” asused herein generally refers to a cell which has been transfected withan exogenous DNA sequence. Cells from the stable human cell line, 293(readily available through, e.g., the American Type Culture Collectionunder Accession Number ATCC CRL1573) are preferred in the practice ofthe present invention. Particularly, the human cell line 293 is a humanembryonic kidney cell line that has been transformed with adenovirustype-5 DNA fragments (Graham et al. (1977) J. Gen. Virol. 36:59), andexpresses the adenoviral E1a and E1b genes (Aiello et al. (1979)Virology 94:460). The 293 cell line is readily transfected, and providesa particularly convenient platform in which to produce rAAV virions.

[0083] AAV Helper Functions

[0084] Host cells containing the above-described AAV expression vectorsmust be rendered capable of providing AAV helper functions in order toreplicate and encapsidate the nucleotide sequences flanked by the AAVITRs to produce rAAV virions. AAV helper functions are generallyAAV-derived coding sequences which can be expressed to provide AAV geneproducts that, in turn, function in trans for productive AAVreplication. AAV helper functions are used herein to complementnecessary AAV functions that are missing from the AAV expressionvectors. Thus, AAV helper functions include one, or both of the majorAAV ORFs, namely the rep and cap coding regions, or functionalhomologues thereof.

[0085] The Rep expression products have been shown to possess manyfunctions, including, among others: recognition, binding and nicking ofthe AAV origin of DNA replication; DNA helicase activity; and modulationof transcription from AAV (or other heterologous) promoters. The Capexpression products supply necessary packaging functions. AAV helperfunctions are used herein to complement AAV functions in trans that aremissing from AAV vectors.

[0086] The term “AAV helper construct” refers generally to a nucleicacid molecule that includes nucleotide sequences providing AAV functionsdeleted from an AAV vector which is to be used to produce a transducingvector for delivery of a nucleotide sequence of interest. AAV helperconstructs are commonly used to provide transient expression of AAV repand/or cap genes to complement missing AAV functions that are necessaryfor lytic AAV replication; however, helper constructs lack AAV ITRs andcan neither replicate nor package themselves. AAV helper constructs canbe in the form of a plasmid, phage, transposon, cosmid, virus, orvirion. A number of AAV helper constructs have been described, such asthe commonly used plasmids pAAV/Ad and pIM29+45 which encode both Repand Cap expression products. See, e.g., Samulski et al. (1989) J. Virol.63:3822-3828; and McCarty et al. (1991) J. Virol. 65:2936-2945. A numberof other vectors have been described which encode Rep and/or Capexpression products. See, e.g., U.S. Pat. No. 5,139,941.

[0087] By “AAV rep coding region” is meant the art-recognized region ofthe AAV genome which encodes the replication proteins Rep 78, Rep 68,Rep 52 and Rep 40. These Rep expression products have been shown topossess many functions, including recognition, binding and nicking ofthe AAV origin of DNA replication, DNA helicase activity and modulationof transcription from AAV (or other heterologous) promoters. The Repexpression products are collectively required for replicating the AAVgenome. For a description of the AAV rep coding region, see, e.g.,Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol.158:97-129; and Kotin, R. M. (1994) Human Gene Therapy 5:793-801.Suitable homologues of the AAV rep coding region include the humanherpesvirus 6 (HHV-6) rep gene which is also known to mediate AAV-2 DNAreplication (Thomson et al. (1994) Virology 204:304-311).

[0088] By “AAV cap coding region” is meant the art-recognized region ofthe AAV genome which encodes the capsid proteins VP1, VP2, and VP3, orfunctional homologues thereof. These Cap expression products supply thepackaging functions which are collectively required for packaging theviral genome. For a description of the AAV cap coding region, see, e.g.,Muzyczka, N. and Kotin, R. M. (supra).

[0089] AAV helper functions are introduced into the host cell bytransfecting the host cell with an AAV helper construct either prior to,or concurrently with, the transfection of the AAV expression vector. AAVhelper constructs are thus used to provide at least transient expressionof AAV rep and/or cap genes to complement missing AAV functions that arenecessary for productive AAV infection. AAV helper constructs lack AAVITRs and can neither replicate nor package themselves. These constructscan be in the form of a plasmid, phage, transposon, cosmid, virus, orvirion. A number of AAV helper constructs have been described, such asthe commonly used plasmids pAAV/Ad and pIM29+45 which encode both Repand Cap expression products. See, e.g., Samulski et al. (1989) J. Virol.63:3822-3828; and McCarty et al. (1991) J. Virol. 65:2936-2945. A numberof other vectors have been described which encode Rep and/or Capexpression products. See, e.g., U.S. Pat. No. 5,139,941.

[0090] Both AAV expression vectors and AAV helper constructs can beconstructed to contain one or more optional selectable markers. Suitablemarkers include genes which confer antibiotic resistance or sensitivityto, impart color to, or change the antigenic characteristics of thosecells which have been transfected with a nucleic acid constructcontaining the selectable marker when the cells are grown in anappropriate selective medium. Several selectable marker genes that areuseful in the practice of the invention include the hygromycin Bresistance gene (encoding Aminoglycoside phosphotranferase (APH)) thatallows selection in mammalian cells by conferring resistance to G418(available from Sigma, St. Louis, Mo.). Other suitable markers are knownto those of skill in the art.

[0091] AAV Accessory Functions

[0092] The host cell (or packaging cell) must also be rendered capableof providing non AAV derived functions, or “accessory functions,” inorder to produce rAAV virions. Accessory functions are non AAV derivedviral and/or cellular functions upon which AAV is dependent for itsreplication. Thus, accessory functions include at least those non AAVproteins and RNAs that are required in AAV replication, including thoseinvolved in activation of AAV gene transcription, stage specific AAVmRNA splicing, AAV DNA replication, synthesis of Cap expression productsand AAV capsid assembly. Viral-based accessory functions can be derivedfrom any of the known helper viruses.

[0093] Particularly, accessory functions can be introduced into and thenexpressed in host cells using methods known to those of skill in theart. Commonly, accessory functions are provided by infection of the hostcells with an unrelated helper virus. A number of suitable helperviruses are known, including adenoviruses; herpesviruses such as herpessimplex virus types 1 and 2; and vaccinia viruses. Nonviral accessoryfunctions will also find use herein, such as those provided by cellsynchronization using any of various known agents. See, e.g., Buller etal. (1981) J. Virol. 40:241-247; McPherson et al. (1985) Virology147:217-222; Schlehofer et al. (1986) Virology 152:110-117.

[0094] Alternatively, accessory functions can be provided using anaccessory function vector. Accessory function vectors include nucleotidesequences that provide one or more accessory functions. An accessoryfunction vector is capable of being introduced into a suitable host cellin order to support efficient AAV virion production in the host cell.Accessory function vectors can be in the form of a plasmid, phage,transposon or cosmid. Accessory vectors can also be in the form of oneor more linearized DNA or RNA fragments which, when associated with theappropriate control elements and enzymes, can be transcribed orexpressed in a host cell to provide accessory functions. See, forexample, International Publication No. WO 97/17548, published May 15,1997.

[0095] Nucleic acid sequences providing the accessory functions can beobtained from natural sources, such as from the genome of an adenovirusparticle, or constructed using recombinant or synthetic methods known inthe art. In this regard, adenovirus-derived accessory functions havebeen widely studied, and a number of adenovirus genes involved inaccessory functions have been identified and partially characterized.See, e.g., Carter, B. J. (1990) “Adeno-Associated Virus HelperFunctions,” in CRC Handbook of Parvoviruses, vol. I (P. Tijssen, ed.),and Muzyczka, N. (1992) Curr. Topics. Microbiol and Immun. 158:97-129.Specifically, early adenoviral gene regions E1 a, E2a, E4, VAI RNA and,possibly, E1b are thought to participate in the accessory process. Janiket al. (1981) Proc. Natl. Acad. Sci. USA 78:1925-1929.Herpesvirus-derived accessory functions have been described. See, e.g.,Young et al. (1979) Prog. Med. Virol. 25:113. Vaccinia virus-derivedaccessory functions have also been described. See, e.g., Carter, B. J.(1990), supra., Schlehofer et al. (1986) Virology 152:110-117.

[0096] As a consequence of the infection of the host cell with a helpervirus, or transfection of the host cell with an accessory functionvector, accessory functions are expressed which transactivate the AAVhelper construct to produce AAV Rep and/or Cap proteins. The Repexpression products excise the recombinant DNA (including the DNA ofinterest) from the AAV expression vector. The Rep proteins also serve toduplicate the AAV genome. The expressed Cap proteins assemble intocapsids, and the recombinant AAV genome is packaged into the capsids.Thus, productive AAV replication ensues, and the DNA is packaged intorAAV virions.

[0097] Following recombinant AAV replication, rAAV virions can bepurified from the host cell using a variety of conventional purificationmethods, such as CsCl gradients. Further, if infection is employed toexpress the accessory functions, residual helper virus can beinactivated, using known methods. For example, adenovirus can beinactivated by heating to temperatures of approximately 60° C. for,e.g., 20 minutes or more. This treatment effectively inactivates onlythe helper virus since AAV is extremely heat stable while the helperadenovirus is heat labile.

[0098] The resulting rAAV virions are then ready for use for DNAdelivery to the CNS (e.g., cranial cavity) of the subject.

Delivery of Viral Vectors

[0099] Methods of delivery of viral vectors include, but are not limitedto, intra-arterial, intra-muscular, intravenous, intranasal and oralroutes. Generally, rAAV virions may be introduced into cells of the CNSusing either in vivo or in vitro transduction techniques. If transducedin vitro, the desired recipient cell will be removed from the subject,transduced with rAAV virions and reintroduced into the subject.Alternatively, syngeneic or xenogeneic cells can be used where thosecells will not generate an inappropriate immune response in the subject.

[0100] Suitable methods for the delivery and introduction of transducedcells into a subject have been described. For example, cells can betransduced in vitro by combining recombinant AAV virions with CNS cellse.g., in appropriate media, and screening for those cells harboring theDNA of interest can be screened using conventional techniques such asSouthern blots and/or PCR, or by using selectable markers. Transducedcells can then be formulated into pharmaceutical compositions, describedmore fully below, and the composition introduced into the subject byvarious techniques, such as by grafting, intramuscular, intravenous,subcutaneous and intraperitoneal injection.

[0101] For in vivo delivery, the rAAV virions will be formulated intopharmaceutical compositions and will generally be administeredparenterally, e.g., by intramuscular injection directly into skeletal orcardiac muscle or by injection into the CNS.

[0102] However, since conventional methods such as injection have notbeen shown to provide widespread delivery of viral vectors to the brainof the subject, central to the present invention is the discovery thatviral vectors are efficiently delivered to the CNS viaconvection-enhanced delivery (CED) systems. The present inventors arethe first to describe and demonstrate that CED can efficiently deliverviral vectors, e.g., AAV, over large regions of an animal's brain (e.g.,striatum). As described in detail and exemplified below, these methodsare suitable for a variety of viral vectors, for instance AAV vectorscarrying reporter genes (e.g., thymidine kinase (tk)) or therapeuticgenes (e.g., AADC and tk).

[0103] Any convection-enhanced delivery device may be appropriate fordelivery of viral vectors. In a preferred embodiment, the device is anosmotic pump or an infusion pump. Both osmotic and infusion pumps arecommerically available from a variety of suppliers, for example AlzetCorporation, Hamilton Corporation, Aiza, Inc., Palo Alto, Calif.).Typically, a viral vector is delivered via CED devices as follows. Acatheter, cannula or other injection device is inserted into CNS tissuein the chosen subject. In view of the teachings herein, one of skill inthe art could readily determine which general area of the CNS is anappropriate target. For example, when delivering AAV-AADC to treat PD,the striatum is a suitable area of the brain to target. Stereotacticmaps and positioning devices are available, for example from ASIInstruments, Warren, Mich. Positioning may also be conducted by usinganatomical maps obtained by CT and/or MRI imaging of the subject's brainto help guide the injection device to the chosen target. Moreover,because the methods described herein can be practiced such thatrelatively large areas of the brain take up the viral vectors, fewerinfusion cannula are needed. Since surgical complications are related tothe number of penetrations, the methods described herein also serve toreduce the side effects seen with conventional delivery techniques.

[0104] Pharmaceutical compositions will comprise sufficient geneticmaterial to produce a therapeutically effective amount of the protein ofinterest, i.e., an amount sufficient to reduce or ameliorate symptoms ofthe disease state in question or an amount sufficient to confer thedesired benefit. The pharmaceutical compositions will also contain apharmaceutically acceptable excipient. Such excipients include anypharmaceutical agent that does not itself induce the production ofantibodies harmful to the individual receiving the composition, andwhich may be administered without undue toxicity. Pharmaceuticallyacceptable excipients include, but are not limited to, sorbitol,Tween80, and liquids such as water, saline, glycerol and ethanol.Pharmaceutically acceptable salts can be included therein, for example,mineral acid salts such as hydrochlorides, hydrobromides, phosphates,sulfates, and the like; and the salts of organic acids such as acetates,propionates, malonates, benzoates, and the like. Additionally, auxiliarysubstances, such as wetting or emulsifying agents, pH bufferingsubstances, and the like, may be present in such vehicles. A thoroughdiscussion of pharmaceutically acceptable excipients is available inREMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. 1991).

[0105] As is apparent to those skilled in the art in view of theteachings of this specification, an effective amount of viral vectorwhich must be added can be empirically determined. Administration can beeffected in one dose, continuously or intermittently throughout thecourse of treatment. Methods of determining the most effective means anddosages of administration are well known to those of skill in the artand will vary with the viral vector, the composition of the therapy, thetarget cells, and the subject being treated. Single and multipleadministrations can be carried out with the dose level and pattern beingselected by the treating physician.

[0106] It should be understood that more than one transgene could beexpressed by the delivered viral vector. Alternatively, separatevectors, each expressing one or more different transgenes, can also bedelivered to the CNS as described herein. Furthermore, it is alsointended that the viral vectors delivered by the methods of the presentinvention be combined with other suitable compositions and therapies.For instance, as described in detail in the Examples below, Parkinson'sdisease can be treated by co-administering an AAV vector expressing AADCinto the CNS (e.g., into the caudate nucleus or putamen of the striatum)and additional agents, such as dopamine precursors (e.g., L-dopa),inhibitors of dopamine synthesis (e.g., carbidopa), inhibitors ofdopamine catabolism (e.g., MaOB inhibitors), dopamine agonists orantagonists can be administered prior or subsequent to or simultaneouslywith the vector encoding AADC. For example, L-dopa and, optionally,carbidopa, may be administered systemically. In this way, the dopaminewhich is naturally depleted in PD patients, is restored, apparently byexpression of AADC which is able to convert L-dopa into dopamine. Wherethe transgene (e.g., AADC) is under the control of an induciblepromoter, certain systemically-delivered compounds such as muristerone,ponasteron, tetracyline or aufin may be administered in order toregulate expression of the transgene.

Treatment of CNS Disorders

[0107] Viral vectors expressing therapeutic transgenes can be used totreat various CNS disorders by providing therapeutic proteins orpolypeptides. In a preferred embodiment, the viral vectors are deliveredto the CNS via the CED methods described herein as these methods providethe first effective way of broadly distributing viral vectors into theCNS. Non-limiting examples of disorders which may be treated includetumors, injury resulting from stroke and neurodegenerative diseases.

[0108] Parkinson's disease

[0109] In a preferred embodiment of the present invention, viral vectorswhich provide the enzyme AADC are used for the treatment of Parkinson'sdisease. As described above, Parkinson's disease results from aselective loss of dopaminergic nigrostriatal neurons, resulting in aloss of input from the substantia nigra to the striatum. Animal modelsof PD have been created, for instance by treating rats or primates with6-hydroxydopamine (6-OHDA) to destroy dopaminergic cells or by lesioningprimates with the neurotoxin1-methyl-4-phenyl-1,2,3,4-tetrahydropyridine (MPTP), which produces aParkinson's-like disease.

[0110] The present invention provides the first evidence thatdopaminergic activity can be restored in Parkinson's patients (e.g.,MPTP-lesioned monkeys), by administration of viral vectors carrying thetransgene for AADC in combination with systemic (e.g., oral)administration of L-dopa and, optionally, carbidopa. Previoussuggestions for gene therapy for Parkinson's have focused on thedeficiency of tyrosine hydroxylase as the disease progresses. Thesesuggestions, therefore, call for the restoration of dopamine synthesisin the nigrostriatal pathway by the successful expression of at leastthree transgenes in order to make dopamine in situ: the tyrosinehydroxylase gene; the gene for the co-factor bioptrene,GTP-cyclohydroxylase-1; and the gene for AADC. In addition to theproblems associated with delivering three genes at appropriate levels,the regulation of dopamine levels would be difficult to control usingthis approach.

[0111] The present inventors have demonstrated that one transgene (e.g.AADC) in combination with L-dopa provides therapeutic benefit. AADC isthe enzyme involved in the final step of dopamine biosynthesis,converting L-dopa to dopamine. Thus, a clear advantage of the AADCtherapeutic approach to restoring dopaminergic activity is that only onegene has to be delivered and the regulation of dopamine levels ispossible by controlling peripheral levels of L-dopa. Furthermore, bydelivering just the AADC gene, L-dopa can be used as a pro-drug toregulate levels of dopamine in the striatum.

[0112] Since AADC-encoding nucleotides delivered by AAV vectors appearto be expressed mainly in the striatal neurons another importanttherapeutic advantage is the treatment's provision of a bufferingmechanism for L-dopa. Many side effects, such as dyskinesisas, areattributed to the inefficient buffering of Parkinsonian brain. Themethods described herein avoid this problem by allowing un-metabolizedL-dopa to be stored in the neurons. As exemplified below, the deliveryof AADC to the MPTP-treated striatum enables conversion of L-dopa todopamine and the subsequent metabolism to DOPAC and HVA by striatalneurons. Based on FMT PET data, it appears that striatal neurons canalso store dopamine, since FMT was visualized in this region. In fact,conversion rates of L-dopa to dopamine following AADC gene transfer wereas robust and greater than seen in the normal striatum (see, e.g., FIG.7). Furthermore, although Parkinson's disease is an progressivedisorder, it is not likely that an ongoing degeneration process willaffect AADC expression in striatal neurons since they are not typicallyaffected by idopathic Parkinson's disease.

[0113] The degeneration of the dopaminergic system in patients withidiopathic Parkinson's disease is not uniform. The nigrostriatal pathwaydegenerates at much faster rate than mesolimbic pathway, leavingpatients with an imbalance between the activity of the two pathways. Asthe disease progresses, higher levels of L-dopa are needed to compensatefor the degeneration of the nigrostriatal pathway, but this also resultsin increasingly higher dopamine levels in the nucleus accumbens andother parts of the mesolimbic system. Such overstimulation may beresponsible for some of the side effects associated with L-dopatreatment such as hallucinations. Similarly, MPTP leaves the mesolimbicdopaminergic system relatively spared (see, FIG. 7, no dopaminergicinnervation is present in the caudate and putamen, a partial lesion isseen in the nucleus accumbens). As shown in FIG. 7, AAV-AADC can restorethis imbalance almost back to normal, therefore, it is possible thatlower doses of L-dopa will be required following the restoration of AADCenzyme levels and improved L-dopa to dopamine conversion rates. This inturn might reduce overstimulation of the mesolimbic system, resulting infewer L-dopa/carbidopa related side effects.

[0114] As explained above, the AAV-AADC vectors can be delivered by anysuitable method, for example, injection, grafting, infusion,transplantation of cells carrying the vectors, etc. In a preferredembodiment, the vectors are delivered by the CED methods describedherein. As exemplified below, such delivery methods provide broaddistribution and expression in CNS neurons and thereby provide a noveltreatment regime for PD.

Imaging

[0115] The present invention also provides methods of determining invivo activity of an enzyme or other molecule. More specifically, atracer which specifically tracks the targeted activity is selected andlabeled. In a preferred embodiment, the tracer tracks dopamine activity,for example fluoro-L-m-tyrosine (FMT) which binds to cells that utilizedopamine. Suitable labels for the selected tracer include anycomposition detectable by spectroscopic, photochemical, immunochemical,electrical, optical or chemical means. Useful labels in the presentinvention include radiolabels (e.g., ¹⁸F, ³H, ¹²⁵I, ³⁵S, ³²P, etc),enzymes, colorimeteric labels, fluorescent dyes, and the like. In apreferred embodiment, the label ¹⁸F is used with FMT to quantifydopamine activity.

[0116] Means of detecting labels are well know to those of skill in theart. For example, radiolabels may be detected using imaging techniques,photographic film or scintillation counters. In a preferred embodiment,the label is detected in vivo in the brain of the subject by imagingtechniques, for example positron emission tomography (PET). PETtechniques are discussed in detail in Example 3 below.

EXAMPLES

[0117] Below are examples of specific embodiments for carrying out thepresent invention. The examples are offered for illustrative purposesonly, and are not intended to limit the scope of the present inventionin any way.

[0118] Efforts have been made to ensure accuracy with respect to numbersused (e.g., amounts, temperatures, etc.), but some experimental errorand deviation should, of course, be allowed for.

Example 1

[0119] CONSTRUCTION AND PRODUCTION OF AAV-TK

[0120] The AAV-tk vector was constructed by placing the herpes simplexvirus thymidine kinase (tk) gene under the transcriptional control ofthe cytomegalovirus (CMV) immediate early promoter in a pUC-basedplasmid (available from, Roche Molecular Biochemicals). A β-globinintron was located directly upstream from the tk gene and human growthhormone poly-A was placed downstream. The entire cassette was flanked byAAV inverted terminal repeats (ITRs) that are required for geneexpression, replication, and packaging into viral particles.

[0121] Recombinant AAV virions were produced in human 293 cells (readilyavailable through, e.g., the American Type Culture Collection underAccession Number ATCC CRL1573) as follows. The 293 cell line wascultured in complete DMEM (Biowhittaker) containing 4.5 g/liter glucose,10% heat-inactivated fetal calf serum (FCS; Hyclone), and 2 mMglutamine. Subconfluent 293 cells were co-transfected by calciumphosphate precipitation (see, e.g., Sambrook, et al.) with the AAV-tkexpression cassette flanked by ITRs and helper plasmids derived frothboth AAV (pw1909, containing the AAV rep and cap genes) and adenovirus(pLadenol, containing E2a, E4, and adenoviral VA₁ and VA₁₁ RNA genes).After 6 hours, the media was changed to DMEM without serum andincubation was continued at 37° C. in 5% CO₂ for 72 hours. Pelletedcells were lysed in Tris buffer (100 mM Tris/150 mM NaCl, pH 8-0) bythree cycles of freeze/thaw, and lysate was clarified of cell debris bycentrifugation at 10,000 g for 15 m. To pellet non-viral protiens, theclarified lysate was centrifuged at 10,000 g for 15 min after addingCaCl₂ to a final concentration of 25 mM and incubated for 1 h at 0° C.Polyethylene glycol 8000 (PEG) was added to the resulting supernatant(final concentration=8%); this solution was incubated for 3 h at 0° C.and centrifuged at 3000×g for 30 minutes. The vector-containing pelletwas solubilized in 50 mM Hepes Na/150 mM NaCl/25 mM EDTA, pH 8.0, andcentrifuged at 10,000×g for 15 minutes to pellet and remove insolublematerial.

[0122] Cesium chloride isopycnic gradient centrifugation was performedand AAV-tk was recovered from the resulting gradient by isolating thefractions with in average density of 1.38 g/ml. PEG was again used toconcentrate vector, which was then resuspended in 25 mM Hepes Na/150 mMNaCl, pH 7.4 and centrifuged as described to remove insoluble material.The stock was treated with DNAse and vector titer was determined byquantitative dot-blot hybridization.

Example 2

[0123] IN VIVO DELIVERY OF AAV-TK: DOSAGES AND METHODS

[0124] In order to determine the appropriate dose of AAV to introduceinto the brain, the following study was conducted. The striatum was usedto test dose response to the AAV vector because of its relatively largearea of homogenous tissue and because it is a target for treatment ofneurodegenerative disease and other central nervous system disorders.

[0125] In addition, efficient methods of delivering vector to the CNSwere determined. Simple stereotactic injection of therapeutic agents hasbeen shown to result in limited volume of distribution in brain (Krollet al, (1996) Neurosurgery 38:746-754). Therefore, slow infusion pumpswere used to maintain a pressure gradient during intracranial delivery.Previous studies concerned with the delivery of small, medium, and largemolecules to brain have demonstrated that slow infusion pump results inextensive and homogenous tissue distribution.

[0126] To investigate which method of administering intracranialinjection of the vector is most efficient, rats were given 2.5×10¹⁰particles of AAV-tk by using the Harvard infusion pump (HarvardApparatus Inc., Holliston, Mass.) or Alzet subcutaneous osmotic pumps(Alza Scientific Products, Palo Alto, Calif.). Female Sprague-Dawleyrats (250-300 g) from Charles River Laboratories (Wilmington, Mass.)were anesthetized with an intraperitoneal injection of ketamine (100mg/kg body weight) and xylazine (10 mg/kg, body weight) and prepped forsurgery. During surgery, sedation was maintained with isofluorane(Attrane, Omeda PPD Inc., Liberty, N.J.) and O₂ flow rates were kept at0.3-0.5 L/m. The head of each rat was fixed in a stereotactic apparatus(Small Animal Stereotactic Frame; ASI Instruemnts, Warren, Mich.) withear bars, and a midline incision was made through the skin to expose thecranium. A bore hole was made in the skull 1 mm anterior to the bregmaand 2.6 mm lateral to the midline using a small dental drill. Vector wasdelivered to the left hemisphere and a depth of 5 mm using an infusionpump or subcutaneous osmotic pumps.

[0127] For dosage studies, there were 3 groups of animals with 6 animalsper group. AAV-tk was continually administered to each rat at a rate of8 μl/h for 2.5 h using a Harvard infusion pump. The loading chamber(Teflon tubing {fraction (1/16)}th″ OD×0.03″ ID) and attached infusionchamber ({fraction (1/16)}″ OD×0.02″ ID) were filled with 2.5×10⁸,2.5×10⁹, or 2.5×10¹⁰ particles of AAV-tk in a total volume of 20 μlartificial csf (148 mM NaCl, 3 mM KCl, 1.4 mM CaCl₂.2H₂0, 0-8 mMMgCl₂.6H₂O, 1.3 mM Na₂HPO₄.H₂O, 0.2 mM Na₂HPO₄.H₂O). Delivery wasthrough a 27 gauge needle fitted with fused silica, which was graduallyremoved 15 m following infusion.

[0128] Alternatively, subcutaneous osmotic pumps were used to delivervector to one group of 6 animals. AAV-tk was continually administered toeach rat at a rate of 8 μl/hour for 24 h using Alzet osmotic pumps,model #2001D (ALZA Scientific Products, Palo Alto, Calif.). The pump'sreservoir and attached catheter (polyethylene 60 tubing) were filledwith 2.5×10¹⁰ particles of AAV-tk in a total volume of 200 μl artificialcsf (Harvard Apparatus, Inc., Holliston, Mass.) Delivery was through a27 gauge cannula fitted with fused silica. After stereotactic placement,the cannula was secured to the skull with a small stainless steel screwand dental cement, and the pump was implanted subcutaneously in themid-scapular area of the back. The surgical site was closed inanatomical layers with 9 mm wound clips. Twenty four hours later, pumpswere removed by clipping and sealing the catheters but the implantedcannulas were left in place. Burr holes were filled with bone wax.

[0129] All surgical procedures, animal care and housing, and tissueharvest were performed at the Richmond facility of the Berkeley AntibodyCo. (Berkeley, Calif.).

Histology

[0130] Animals were euthanized by pentobarbitol overdose (dose) andperfused through the ascending aorta with ice-cold PBS and 4% neutralbuffered paraformaldehyde. The brains were removed from the skull,post-fixed by immersion in the same fixative for 24 hr, equilibrated in30% sucrose, and frozen in −70° C. isopentane. They were then positionedin a cryostat and 40 micron sections were serially collected from theprefrontal cortex to the midbrain. Each brain yielded approximately 150sections, that spanned an anterior-posterior (AP) length of 6-8 mm. Forroutine histological analysis, every IP section was stained with H & E.Selected sections representing different brain regions were stained withcrystal violet in order to determine general cell density. Results areshown in Table 1. TABLE 1 Cellular Infilt. Necrosis Bleeding FreshHemosiderin Track Needle Infusion Pump (2 × 10⁸) − − − − Fine linesInfusion Pump (2 × 10⁹) − − + − Fine lines Infusion Pump (2 × 10¹⁰) − ++(1/6) ++ (4/6) − Fine lines Osmotic Pump (2 × 10¹⁰) +++ (5/6) +++ ++ +++Holes

PCR Analysis

[0131] Two additional groups of rats, with two animals per group, weretreated with 2.5×10¹⁰ particles AAV-tk for the purpose of determiningtissue distribution of vector. One of the groups received vector byinfusion and the other by osmotic pump, as described above. Animals wereeuthanized three weeks later using C0₂ inhalation and samples from 15organs and tissues were harvested from each rat including right brain,left brain, spinal cord, right eye, left eye, heart, lung, liver,kidney, spleen, ovary, thymus, lymph node, bone marrow, and leg muscle.Sterile techniques were used and tissue was collected using disposablesuture removal kits that were changed between each sample. Tissues wereimmediately frozen in liquid N₂ and kept at −70° C. until they wereprocessed for genomic DNA. PCR was performed using Perkin Elmer'sGeneAmp PCR Core Kit and two 30-mer oligos derived from tk sequence(5′-AAGTCATCGGCTCGGGTACGTAGACGATATC-3′ (SEQ ID NO:1) and5′ATAGCAGCTACAATCCAGCTACCATTCTGC-3′ (SEQ ID NO:2)). Reactions wereperformed in a PTC-100 thermal cycler (M J Research, Inc.) and resultedin a 158 bp per product in samples where vector was present.

Immunohistochemisty

[0132] Immunocytochemistry was used to detect transgene expression inevery section that directly followed one stained with H & E. Thus, oneout of every 12 sections was washed in PBS, treated with 3% H₂O for 30 mto block endogenous peroxidase activity, rinsed again in dH₂0 and PBS,and incubated in blocking solution (10% goat serum+0.01% Triton-X100 inPBS) for 30 m. Next, samples were incubated in polygonal anti-tkantibody (Yale) (1:1000) for 1 h, washed three times in PBS, incubatedin biotinylated goat anti-rabbit IgG (Vector) (1:300) for 1 h, andwashed again. Antibody binding was visualized with Streptavidinhorseradish peroxidase (1:300) and VIP chromogen (Vector).

Quantitative Analysis

[0133] Transgene expression was quantitated for each brain by using aKen-a-vision microprojector to project tk-immunostained sections onto anARTZII graphic tablet. The NIH image 1.6 program was used to capture andanalyze images. The total estimated number of positive cells for eachbrain was determined at a magnification of 100× using the followingformula:

[0134] Total tk cell#=(n1+n2+n3 . . . )×12×k

[0135] n=positive cells/section

[0136] k: correction factor derived from the Ambercrombie equation(1946): k=T/(T+D)

[0137] T=section thickness (40 μ), D cell diameter (16 μ); k=0.71

[0138] The volumes of the tk-immunoreactive regions were determined bymeasuring areas of tk expression using captured images projected at 5O×magnification and calculating as follows:$A_{x} = {{{average}\quad {area}\quad \left( {mm}^{2} \right)} = \frac{{a\quad 1} + {a\quad 2} + {a\quad 3\quad \ldots \quad {An}}}{n}}$

[0139] (where, L=AP distance (μ) of staining=n×12×40 u and n=# ofstained sections)

[0140] To determine how much virus is required to efficiently transducebrain tissue, comparisons of immunostained sections were performedbetween rats receiving 2.5×10⁸, 2.5×10⁹, or 2.5×10¹⁰ particles of AAV-tkvia the Harvard infusion pump.

[0141] With all parameters measured, a clear dose response was observed.Infused tissue from animals receiving the highest titer demonstratedtransgene expression in an average of 300 mm³ of tissue (orapproximately 60% of an adult rat cerebral hemisphere (Leyden et al.(1998) Behav. Brain Res. 87:59-67) as compared to volumes of 10 mm³ forthe middle-dose group and <1 mm³ for the low-dose group (FIG. 1a).Volumes were calculated from the mean areas and lengths of stainingwhich both also showed significant differences between the groups (FIG.1b,c). Expression within a volume of transduced tissue was not uniform,however, but exhibited a gradient of staining. Areas directlysurrounding the injection sites were heavily labeled while fewerpositive cells could be detected as distance from the needle tractincreased (FIG. 2). Finally, FIG. 1d illustrates that the total numberof tk-positive cells in section, from the high dose group, estimated toaverage 169,000, is approximately 10× higher than that of the middledose group.

Infusion Versus Osmotic Pump Delivery

[0142] Comparisons of immunostained brain sections demonstratedsimilarities and differences in the abilities of the two pumps todeliver vector. There was no significant difference in tk expressionbetween the two groups as measured by mean volume, area, AP distance,and total estimated number of positive cells (FIG. 3a-d). Because therewas some tissue loss surrounding the needle tracts of all of the sampleswithin the osmotic pump delivery group (data not shown), that group'strue mean value for the number of estimated positive cells may behigher. And, while both delivery methods resulted in notable transgeneexpression, there was a difference in the type of cells that becamelabeled. Tissue infused with vector expressed tk almost exclusively inneurons (FIG. 4a,b) and tissue receiving vector via osmotic pumpexhibited expression in neurons and in reactive glial cells close to thesite of injection (FIG. 4c,d).

Tissue Distribution

[0143] To determine if recombinant AAV could be detected at locationsdistant from the site of intracranial delivery, PCR analysis wasperformed on 15 different organs and tissue from each of three rats whohad received a high dose of vector. Regardless of the delivery method(infusion or osmotic pumps), a 458 bp PCR product from the tk gene couldbe detected in spinal cord, spleen, and both hemispheres of the brainusing Southern blot analysis (FIG. 6). In one of the rats, vector setswere also detected in tissue from the kidney.

Toxicity

[0144] To assess whether or not toxicity was associated with any of thedelivery methods, histopathology was performed on H & E sections fromeach group and the results are summarized in table 1. Overall tissuemorphology was well preserved and no freezing or other artifacts werepresent. In the infusion delivery groups, tissue damage was minimal, ifpresent at all. There was no cellular infiltration, no necrosis in theneedle tract, and minimal cortical necrosis in a few of the animals.Fresh bleeding was found in one of the high-dose rats, andhemosiderosis, indicating moderate bleeding in the past, was found infour of the high-dose animals. Alternatively, serious damage was notedin all of the animals in the osmotic pump delivery group including largenecrotic areas surrounding the needle tract, cellular infiltrates, andhemosiderosis.

[0145] Thus, infusion of 2.5×10¹⁰ particles of AAV-tk at 8 μl/h for 2.5h is sufficient to partially distribute AAV vector to a volume of 300mm³ of tissue. Within this region, a gradient of expression is observedwith heavy staining directly surrounding the site of injection and fewerpositive cells farther away. Distribution appears to be a function ofdose (particle number) and not a function of delivery time or samplevolume when two different pump delivery systems are compared: thedistribution of 2.5×10¹⁰ particles was the same whether it was deliveredby osmotic pump (volume=200 μl, rate=8 μl/h, time=24 h) or infusion pump(volume=20 μl, rate=8 μl/h, time=2.5 h). Furthermore, strikinglydifferent levels of expression were observed between the three infusiondelivery groups, where sample volume, rate, and delivery time were keptconstant and particle number was the only variable.

[0146] These results obtained with convection-enhanced delivery of AAVare consistent with those obtained from CED studies of largemacromolecules, such as supramagnetic particles to rat brains. (Kroll etal. (1996) Neurosurg. 38:746-754, U.S. Pat. No.5,720,720). Kroll et al.used magnetic resonance imaging and histochemical staining demonstratedthat dose was the most important variable in maximizing the distributionof particles in tissue. Kroll reports that regardless of whether theinfusion volume was small (2 μl) or moderate (24 μl) or whether theinfusion rate was low (6 μl/h) or high (72 μl/h), increasing theparticles from 5.3 to 26.5 μg resulted in as much as a 5-fold increasein the volume of their distribution in tissue.

[0147] Concerning cell-type specificity, it has been previously reportedthat AAV is capable of transducing neurons and the present studyconfirms this finding. The fact that expression was so prominent inneurons suggests that AAV gene therapy vectors employing the CMVpromoter are useful for treatment of neurodegenerative diseases such asParkinson's and Alzheimer's disease. No expression was seen in matureglial cells, except in small areas of disturbed tissue where activegliosis was present. However, we have previously demonstrated that theAAV-CMV-tk vector is expressed well in glioma cells and, when given inconjunction with the prodrug ganciclovir, is effective in treatingexperimental gliomas in nude mice. Because the tk “suicide” gene isthought to be toxic to dividing cells, it should pose a risk only to thetargeted tumor cells and not to surrounding neurons. Finally, while theCMV promoter used in this study allows for strong transgene expressionin neurons, the choice of cell-type-specific promoters will allowtargeting of AAV to other CNS components such as oligodendrocytes andglial calls.

[0148] The present study also shows that AAV delivered to brain iscontained mostly in the central nervous system. Others have demonstratedretrograde transport of viruses between the two hemispheres of brain andability of viruses to reach spinal cord via circulating cerebral spinalfluid. The appearance of vector in the spleen is curious, and suggests acouple of mechanisms. One is that virus enters the bloodstream duringthe infusion process and circulated through the spleen where it is“scavenged”. If this were the case, however, other tissues that havebeen shown to be inducible by AAV would be expected to also take upvirus. Another possible mechanism could be one exhibited by dendriticcells. These cells found mostly in skin, take up foreign material, enterthe circulation, and concentrate in the spleen where the foreign mattercan exist for long periods of time awaiting further processing ordestruction. In any case, we have found that regardless of the route ofdelivery, including intramuscular, intravenous, and now intracerebral,vector is always detected in the spleen.

[0149] In summary, slow intracranial infusion of high doses of AAVvector has been shown to transduce a significant portion or brain in arodent model. AAV may be used to target a myriad of central nervousdisorders, including tumors, injury resulting from stroke, andneurodegenerative disease.

Example 3

[0150] GENE THERAPY OF PARKINSON'S DISEASE

[0151] Convection-enhanced delivery of AAV vectors carrying thetransgene encoding AADC was shown to restore dopaminergic systems inMPTP-induced Parkinson's disease in monkeys as follows.

Animals

[0152] Rhesus monkeys (n=4, 3-5 kg) were chosen as candidates forimplantation based on the evolution of their parkinsonian symptoms.Animals were lesioned by infusing 2.5-3.5 mg of MPTP-HLC through theright internal carotid artery ( referred to as ipsilateral side)followed by 4 I.V. doses of 0.3 mg/kg of MPTP-HCL (referred to ascontralateral side) until a stable, overlesioned hemi-parkinsoniansyndrome was achieved (Eberling, (1998) Brain Res. 805:259-262). Theprimate MPTP model is considered the gold standard model of evaluationprior to human trials. (Langston (1985) Trends Pharmcol. Sci.6:375-378). MPTP is it converted in the CNS to MPP+ by monoamine oxidaseB. MPP+ is a potent neurotoxin which causes degeneration of the nigraldopaminergic neurons and loss of the nigro-striatal dopamine pathway, asseen in Parkinson's disease. MPTP-lesioned animals were clinicallyevaluated once a week using a clinical rating scale and activitymonitoring for 5 months prior to surgery.

[0153] Following MPTP administration, the animals developed clinicalsigns of Parkinson's disease manifested by general slowness,bradykinesia, rigidity, balance disturbances, and flexed posture. Theleft arm was less frequently used than the right in all of the monkeys,and all showed signs of tremor. Using the clinical rating scale, all ofthe monkeys had moderate to severe stable parkinsonian scores (23±1.7,23±1.2, 24±1.7, 19±3) during the 5 month post-MPTP period.

Vector Production

[0154] 1. pAAV-AADC:

[0155] A 1.5 kb BamHI/PvuII human AADC cDNA (Fan et al.(1998) Human GeneTherapy 9:2527-2535) was cloned into the AAV expression cassette pV4.1cat BamHI/HindII sites. The expression cassette contains a CMV promoter,a chimeric intron composed of a CMV splice donor and a human β-globinsplice acceptor site, human growth hormone polyadenylation sequence, andflanking AAV ITRs (inverted terminal repeats) (Herzog, R. W., et al.(1999) Nature Medicine 5:56-63.).

[0156] 2. pAAV-LacZ:

[0157] The vector pAAV-LacZ was constructed as follows. The AAV codingregion of pSub201 (Samulski et al. (1987) J. Virol 61:3096-3101),between the XbaI sites, was replaced with EcoRI linkers, resulting inplasmid pAS203. The EcoRI to HindIII fragment of pCMVβ (CLONETECH) wasrendered blunt ended and cloned in the Klenow treated EcoRI site ofpAS203 to yield pAAV-lacZ.

[0158] 3. pHLp19:

[0159] Plasmid H19 encodes a modified AAV-2 genome designed to enhancedAAV vector production while suppressing the generation of replicationcompetent pseudo-wild type virus. The plasmid contains a P5 promotermoved to a position 3′ of the cap gene and the promoter is replaced by a5′ untranslated region primarily composed of a FLP recombinaserecognition sequence. pH19 was constructed so as to eliminate anyregions of homology between the 3′ and 5′ ends of the AAV genome.Additionally, the seven base pair TATA box of the pH19 P5 promoter wasdestroyed by mutation of that sequence to GGGGGGG.

[0160] pH19 was constructed in a several step process using AAV-2sequences derived from the AAV-2 provirus, pSM620. pSM620 was digestedwith SmaI and PvuII, and the 4543 bp, rep and cap gene encoding SmaIfragment was cloned into the SmaI site of pUC119 to produce the 7705 bpplasmid, pUCrepcap. The remaining ITR sequences flanking the rep and capgenes were then deleted by oligonucleotide-directed mutagenesis usingthe following oligonucleotides: 145A; 5′-GCT CGG TAC CCG GGC GGA GGG GTGGAG TCG-3′ (SEQ ID NO:3) 145B; 5′-TAA TCA TTA ACT ACA GCC CGG GGA TCCTCT-3′ (SEQ ID NO:4)

[0161] The resulting plasmid, pUCRepCapMutated (pUCRCM) (7559 bp)contains the entire AAV-2 genome without any ITR sequence (4389 bp).SrfI sites, in part introduced by the mutagenic oligonucleotides, flankthe rep and cap genes in this construct. The AAV sequences correspond toAAV-2 positions 146-4,534.

[0162] An Eco47III site was introduced at the 3′ end of the P5 promoterin order to facilitate excision of the P5 promoter sequences. To dothis, pUCRCM was mutagenized with primer P547 (5′-GGT TTG AAC GAG CGCTCG CCA TGC-3′) (SEQ ID NO:5). The resulting 7559 bp plasmid was calledpUCRCM47III.

[0163] The polylinker of pBSIIsk+ was changed by excision of theoriginal with BSSHII and replacement with oligonucleotides blunt 1 and2. The resulting plasmid, bluntscript, is 2830 bp in length and the newpolylinker encodes the restriction sites EcoRV, HpaI, SrfI, PmeI, andEco47III. The blunt 1 and 2 sequences are as follows: blunt 1; 5′-CGCGCC GAT ATC GTT AAC GCC CGG GCG TTT AAA CAG CGC TGG-3′ (SEQ ID NO:6)blunt 2; 5′-CGC GCC AGC GCT GTT TAA ACG CCC GGG CGT TAA CGA TAT CGG-3′(SEQ ID NO:7)

[0164] pH1 was constructed by ligating the 4398 bp, rep and cap geneencoding SmaI fragment from pUCRCM into the SmaI site of pBluntscriptsuch that the HpaI site was proximal to the rep gene. pH1 is 7228 bp inlength.

[0165] pH2 is identical to pH1 except that the P5 promoter of pH1 isreplaced by the 5′ untranslated region of pGN1909. To do this, the 329bp AscI(blunt)-SfiI fragment encoding the 5′ untranslated region frompW1909lacZ was ligated into the 6831 bp SmaI(partial)-SfiI fragment ofpH1 creating pH2. pH2 is 7156 bp in length.

[0166] A P5 promoter was added to the 3′ end of pH2 by insertion of the172 bp, SmaI-Eco43III fragment encoding the p5 promoter from pUCRCM47IIIinto the Eco47III site in pH2. This fragment was oriented such that thedirection of transcription of all three AAV promoters are the same. Thisconstruct is 7327 bp in length.

[0167] The TATA box of the 3′ P5 (AAV-2 positions 255-261, sequenceTATTTAA) was eliminated by changing the sequence to GGGGGGG using themutagenic oligonucleotide 5DIVE2 (5′-TGT GGT CAC GCT GGG GGG GGG GGC CCGAGT GAG CAC G-3′) (SEQ ID NO:8). The resulting construct, pH19, is 7328bp in length.

[0168] 4. pladeno5:

[0169] Pladeno 5 is a plasmid that provides a complete set of adenovirushelper functions for AAV vector production when transfected into 293cells. Essentially, it is composed of the E2A, E4, and VA RNA regionsfrom adenovirus-2 and a plasmid back bone. The plasmid was constructedas follows.

[0170] pBSIIs/k+ was modified to replace the 637 bp region encoding thepolylinker and alpha complementation cassette with a single EcoRV siteusing oligonucleotide directed mutagenesis and the followingoligonucleotide: 5′-CCG CTA CAG GGC GCG ATA TCA GCT CAC TCA A-3′ (SEQ IDNO:9). A polylinker encoding the restriction sites BanHI, KpnI, SrfI,XbaI, ClaI, Bst1107I, SalI, PmeI, and NdeI was then cloned into theEcoRV site (5′-GGA TCC GGT ACC GCC CGG GCT CTA GAA TCG ATG TAT ACG TCGACG TTT AAA CCA TAT G-3′) (SEQ ID NO:10).

[0171] Adenovirus-2 DNA was digested and restriction fragments encodingthe E2A region (a 5,335 bp, KpnI-SrfI fragment corresponding topositions 22,233-27,568 of the adenovirus-2 genome) and the VA RNAs (a731 bp, EcoRV-SacII fragment corresponding to positions 10,426-11,157 ofthe adenovirus-2 genome) were isolated. The E2A fragment was installedbetween the SalI and KpnI sites of the polylinker. An E4 region wasfirst assembled in pBSIIs/k+ by ligating a 13,864 bp, BamHI-AvrIIfragment corresponding to adenovirus-2 positions 21,606-35,470 (encodingthe 5′ end of the gene) and a 462 bp, AvrII and SrfI, digested PCRfragment corresponding to adenovirus-2 positions 35,371-35,833 (encodingthe 3′ end of the gene) between the BamHI and SmaI sites of pBSIIs/k+.The oligonucleotides used to produce the PCR fragment were designed tointroduce a SrfI site at the junction were the E4 promoter and theadenovirus terminal repeat intersect and have the sequences 5′-AGA GGCCCG GGC GTT TTA GGG CGG AGT AAC TTG C-3′ (SEQ ID NO:11) and 5′-ACA TACCCG CAG GCG TAG AGA C-3′ (SEQ ID NO:12). The intact E4 region wasexcised by cleavage with SrfI and SpeI and the 3,189 bp fragmentcorresponding to adenovirus-2 positions 32,644-35,833 was cloned intothe E2A intermediate between the SrfI and XbaI sites. Finally, the VARNA fragment was inserted into the Bst1107 site after T4polymerase-mediated blunt end modification of the SacII site. The genesin pladeno 5 are arranged such that the 5′ ends of the E2A and E4promoters abut, causing the regions to transcribe away from each otherin opposite directions. The VA RNA genes, which are located at the threeprime end of the E4 gene, transcribe towards the E4 gene. The plasmid is11,619 bp in length.

AAV Vector Production

[0172] The HEK 293 cell line (Graham, F. L., Smiley, J., Russel, W. C.,and Naiva, R. (1977) Characteristics of a human cell line transformed byDNA from human adenovirus type 5. J. Gen. Virol. 36:59-72.) was culturedin complete DMEM (BioWhittaker) containing 4.5 g/liter glucose, 10%heat-inactivated fetal calf serum (FCS), and 2mM glutamine at 37° C. in5% CO₂ in air. Forty T225 flasks were seeded with 2.5×10⁶ cells each andgrown for three days prior to transfection to 70-80% confluency(approximately 1.5×10⁶ cells per flask).

[0173] The transfection and purification methods described by Matsushitaet al (Matsushita, T., Elliger, S., Elliger, C., Podsakoff, G.,Villarreal, L., Kurtzman, G. J., Iwaki, Y., and Colosi, P. (1998)“Adeno-associated virus vectors can be efficiently produced withouthelper virus,” Gene Therapy 5:938-945) were employed for AAV vectorproduction, with minor modifications. The vector production processinvolved co-transfection of HEK 293 cells with 20 μg of each of thefollowing three plasmids per flask: the AAV-AADC plasmid, the AAV helperplasmid (pHLP19, containing the AAV rep and cap genes), and theadenovirus helper plasmid (pladeno-5, previously known as pVAE2AE4-2 (4)and composed of the E2A, E4, and VA RNA genes derived from purifiedadenovirus-2), using the calcium phosphate method (Wigler, M. et al.(1980) Transformation of mammalian cells with an amplifiabledominant-acting gene. Proc. Natl. Acad. Sci. USA 77:3567-3570) for aperiod of 6 hrs. After transfection, the media was replaced and thecells were harvested 3 days later. The cell pellets were then subjectedto 3 cycles of freeze-thaw lysis (alternating between dry ice-ethanoland 37° C. baths with intermittent vortexing). The cell debris wasremoved by centrifugation (10,000 g for 15 min). The supernatant wascentrifuged a second time to remove any remaining turbidity andsubsequently treated with Benzonase® (200 u/ml) at 37° C. for 1 hr inorder to reduce contaminating cellular DNA. Following incubation, thesupernatant was made 25 mM in CaCl₂, and was placed on ice for 1 hr. Theresulting precipitate was removed by centrifugation (10,000 g for 15min.) and discarded. The supernatant was then made 10% in PEG(8000), andwas placed on ice for 3 hrs. The precipitate was collected bycentrifugation (3000 g for 30 min) and resuspended in 4 ml of 50 mMNaHEPES, 0.15M NaCl, 25 mM EDTA (pH 8.0) per 20 T225 flasks. Solid CsClwas added to produce a density of 1.4 g/ml and the sample wascentrifuged at 150,000 g for 24 hrs in a Beckman TI70 rotor.AAV-containing fractions were pooled, adjusted to a density of 1.4 g/mlCsCl, and centrifuged at 350,000 g for 16 hrs in a Beckman NVT65 rotor.The fractions containing AAV were then concentrated and diafilteredagainst excipient buffer (5% sorbitol in PBS). The titer of the purifiedAAV-AADC vector was determined using quantitative dot blot analysis andvector stocks were stored at −80° C.

Viral Infusion

[0174] In the surgery room, a sterile field was created to prepare theinfusion system. Infusion cannulae were flushed with saline to assessthe integrity between the needle and tubing interface. Sterile infusioncannulae and loading lines were connected using the appropriate fittingswith extreme caution taken to prevent the collection of air bubbles inthe system. Non-sterile oil infusion lines were prepared as previouslydescribed and 1 m1 gas tight Hamilton syringes filled with oil wereattached to a Harvard infusion pump. Six infusion cannulae were fittedonto microdialysis holders (3 cannulae per holder) and mounted onto astereotactic tower. Following the union of the oil and loading lines,the needle cannulae were primed with AAV and the infusion systemtransferred to the surgery table. Initial infusion rates were set at 0.1p1/min., the lines visually inspected to ensure a smooth flow of fluidthrough the system, and the cannulae manually lowered to their targetsites. A final visual inspection was performed to check for any airbubbles in the infusion system.

[0175] The cannula system consisted of three components: (i) a sterileinfusion cannula; (ii) a sterile loading line housing AAV-AADC orAAV-LacZ (control); and (iii) a non-sterile infusion line containingolive oil. Preparation of each line is described here briefly. Theinfusion cannula consisted of 27 G needles (outer diameter, .03″; innerdiameter, .06″; Terumo Corp., Elkton, Md.) fitted with fused silica(outer diameter, .016″, inner diameter, .008″; Polymicro Technologies,Phoenix, Ariz.), and placed in Teflon tubing (.03″ ID, UpchurchScientific, Seattle, Wash.) such that the distal tip of the silicaextended approximately 15 mm out of the tubing. The needle was securedinto the tubing using superglue and the system was checked for leaksprior to use. At the proximal end of the tubing, a Tefzel fitting andferrule were attached to connect the adjacent loading line.

[0176] Loading and infusion lines consisted of 50 cm sections of Teflontubing (outer diameter, .062″; inner diameter, .03″) fitted with Tefzel{fraction (1/16)}″ ferrules, unions, and male Luer-lock adapters(Upchurch Scientific, Oak Harbor, Wash.) at the distal ends. The sterileloading lines accommodated up to a 1000 ml volume and were primed withsaline prior to use.

[0177] The animals were initially sedated with Ketamine (Ketaset; 10mg/kg, i.m.), intubated and prepped for surgery. A venous line wasestablished using a 22 gauge catheter positioned in the cephalic orsaphenous vein to deliver isotonic fluids at 5-10 ml/kg/hr. Isoflurane(Aerrane, Omeda PPD Inc., Liberty, N.J.) was delivered at 1-3% until theanimal maintained a stable plane of anesthesia. The head was placed inan MRI compatible stereotactic frame according to pre-set valuesattained during a baseline MRI scan. The animal was instrumented withsubcutaneous electrocardiogram electrodes, a rectal probe and the bodycovered with circulating water blankets to maintain a core temperatureof 36-38° C. Electrocardiogram and heart rate (using the Silogic ECG-60,Stewartstown, Pa.) and body temperature were continuously monitoredduring the procedure. The head was prepped with Betadine and 70%ethanol, a sterile field was created and a midline incision performedthrough the skin, muscle and fascia using electrocautery (SurgistatElectrosurgery, Valleylab Inc., Boulder, Colo.).

[0178] Gentle retraction of fascia and muscle allowed for cranialexposure over cortical entry sites. A unilateral craniotomy wasperformed using a Dremel dental drill to expose a 3 cm×2 cm area of duramater above the target sites. Multiple needle cannulae attached to aholder were stereotactically guided to striatal target sites. Surgicalparameters for unilateral infusion of AAV into the hemisphereipsilateral to ICA MPTP infusion are summarized in Table 2. TABLE 2Surgical Parameters for AAV infusion Target Sites Striatum (2 caudate, 4putamen) Hemisphere right side (ipsilateral to ICA MPTP infusion)Infusion Volume 30 μL/site Infusion Rates 0.1 μl/min (60 mm) 0.2 μl/min(60 mm) 0.4 μl/min (30 mm) Virus AAV-AADC; 2.1 × 10e12 particles/ml; Lotno 176.12; 200 μl/vial Control Article AAV-LacZ; 9.2 × 10e11particles/ml; Lot no. 176.126; 200 μl/vial

[0179] Approximately fifteen minutes following infusion, the cannulaeassembly was raised at a rate of 1 mm/min. until it was out of thecortex. The cortex was rinsed with saline, the bone margins trimmed withronguers and the wound site closed in anatomical layers. Analgesics(Numorphan, 1M) and antibiotics (Flocillin, 1M) were administered aspart of the surgical protocol. Animals were monitored for full recoveryfrom anesthesia, placed in their home cages and clinically observed(2×/day) for approximately five days following surgery. Totalneurosurgery time was 4.5 hours per animal.

[0180] Following intrastriatal AAV administration, animals were assessedfor any signs of abnormal behavior. Animals were observed and rated bythe veterinary technicians twice a day using clinical observation forms.All monkeys recovered from the surgery within 2 hours and were able tomaintain themselves, including feeding and grooming. There were no signsof any adverse effects during the entire 8-week post-surgical period.

Magnetic Resonance Imaging

[0181] Visualization of the target site is crucial for the preciseplacement of cells within the caudate nucleus or putamen. Stereotacticprocedures combined with MRI were used in order to accurately place thecannula within the desired targeted structures. All animals were scannedbefore surgery to generate accurate stereotactic coordinates of thetarget implant sites for each individual animal. The same fiducialmarkers that are used for PET scanning were placed on the frame forco-registration of MRI and PET images. Briefly, during the scanningprocedure, the animals were sedated using a mixture of ketamine(Ketaset, 7 mg/kg, im) and xylazine (Rompun, 3 mg/kg, im). The animalswere placed in an MRI compatible stereotactic frame, earbar and eyebarmeasurements were recorded, and an IV line was established. Sixtycoronal images (1 mm) and 15 sagittal images (3 mm) were taken using aGE Signa 1.5 Tesla machine. Magnetic resonance images were Ti-weightedand obtained in three planes using a spoil grass sequence with arepetition time (TR)=700 ms, an echo time (TE)=20 ms and a flip angle of30′). The field of view was 15 cm, with a 192 matrix and a 2 NEX (numberof averages per signal information). Baseline scanning time wasapproximately 20 minutes. Rostro-caudal and medio-lateral distributionof a targeted structure (e.g., caudate nucleus) was determined using thecoronal MR images. Surgical coordinates were determined from magnifiedcoronal images (1.5×) of the caudate nucleus and putamen.

Positron Emission Tomography (PET)

[0182] All 4 animals received 2 PET scans, a baseline scan followingestablishment of the MPTP lesion, and a second scan 7-8 weeks afterinfusion with either AAV-AADC or AAV-LacZ. Prior to PET, each animalunderwent magnetic resonance (MR) imaging using a 1.5 T magnet and astereotaxic frame which permitted coregistration between PET and MR datasets through the use of external fiducial markers. The PET studies wereperformed on the PET-600 system, a singleslice tomograph with aresolution of 2.6 mm in-plane and an adjustable axial resolution whichwas increased from 6 mm to 3 mm for the current study by decreasing theshielding gap. The characteristics of this tomograph have been describedpreviously (Budinger et al. (1991) Nucl. Med. Biol. 23(6):659-667; Valk,(1990) Radiology 176(3):783-790). The monkeys were intubated andanesthetized with isoflurane, placed in a stereotaxic frame andpositioned in the PET scanner so as to image a coronal brain slicepassing through the striatum. Monkeys were positioned in the same wayfor each study using the anterior-posterior scales on the sterootaxicframe and a laser light connected to the tomograph. After beingpositioned in the scanner, a 5 min transmission scan was obtained inorder to correct for photon attenuation, and to check the positioning ofthe animal. The monkeys were then injected with 10-15 mCi of the AADCtracer, 6-[¹⁸F]fluro-L-m-tyrosine (FMT) and imaging began. Imagingcontinued for 60 min, at which time the monkey was repositioned so as toimage a second slice 6 mm caudal to the first.

[0183] The PET and MR datasets were co-registered and regions ofinterest (ROs) were drawn for the striatum in the contralateralhemisphere (the side opposite to ICA MPTP infusion) on PET datacollected at 50 to 60 min (slice 1) and from 65 to 75 min (slice 2) withreference to the MR. Mirror images of the ROs were created in theipsilateral hemisphere (side of MPTP infusion) and radioactivity counts(cm²/sec) were determined for each ROI. Striatal counts were averagedover the two slices for each study. FMT uptake asymmetry ratios werecalculated for each animal at each time point by subtracting the countsfor the ipsilateral (lesioned) striatum from the counts for thecontralateral (un-lesioned) striatum and dividing by the average countsfor the ipsilateral and contralateral striata. In order to reducebetween animal variability in asymmetry ratios, a change score wascalculated by subtracting the asymmetry ratio from the second PET studyfrom the asymmetry ratio for the baseline study for each animal.Unpaired t-tests were used to compare the change in pet asymmetry ratiosfor the AAV-AADC and AAV-LacZ monkeys.

[0184] As expected, all 4 monkeys showed greater FMT uptake in thecontralateral than in the ipsilateral striatum at baseline, which showednegligible uptake. At the time of the second PET study, the AAV-AADCtreated monkeys showed increased FMT uptake in the ipsilateral striatum,while the AAV-LacZ treated animals showed no change from baseline. (FIG.7). The change in FMT uptake asymmetry from baseline to the second PETstudy was significantly (p <0.01) greater for the AAV-AADC monkeys,which showed little asymmetry at the time of the second study, than forthe AAV-LacZ monkeys, which showed greater contralateral FMT uptake atboth time points. (FIG. 8)

Necropsy

[0185] Animals were deeply anesthetized with sodium pentobarbital (25mg/kg i.v.) and sacrificed 8-9 weeks following AAV administration andone week following postsurgical PET scans. On the day of sacrifice,blood samples were taken, and the animals were treated withL-dopa/carbidopa preparation (Sinemet 250/25). Plasma and cervical CSFwere collected and at the time of necropsy. The brains were removed30-45 minutes following the Sinemet administration, placed in the brainmatrix and sectioned coronally into 3-6mm slices. One 3 mm thickstriatal brain slice from each monkey was immediately frozen in −70° C.isopentane and stored frozen for biochemical analysis. The remaining 6mmthick slices were post-fixed in formalin for 72 hours, washed in PBS for12 hrs and adjusted in ascending sucrose gradient (10-20-30%) andfrozen.

Histological Analysis

[0186] The formalin-fixed brain slices were cut into 30 μm thick coronalsections in a cryostat. Frozen sections were collected in seriesstarting at the level of the rostral tip of the caudate nucleus all theway caudally to the level of the substantia nigra. Each section wassaved and kept in antifreeze solution at 70° C. Serial sections werestained for tyrosine hydroxylase (TH), dopa decarboxylase (DDC) orB-galactosidese (B-gal) immunorectivity (IR). Every 12th section waswashed in phosphate buffered saline (PBS) and incubated in 3% H202 for20 min to block the endogenous peroxidase activity. After washing inPBS, the sections were incubated in blocking solution (10% normal horseserum for TH or 10% normal goat serum for DDC and B-gal and 0.1%Triton-XI 00 in PBS) for 30 min, followed by incubation in primaryantibody solution-TH (mouse monoclonal, Chemicon, 1:1000), DDC (rabbitpolygonal, Chemicon, 1:2000) or B-gal (rabbit polygonal, Cortex Blochem,1:5000) for 24 h. The sections were then incubated for 1 h inbiotinylated anti-mouse IgG secondary antibody for TH or anti-rabbit IgGsecondary antibody for DDC and B-gal (Vector Labs, 1:300). The antibodybinding was visualized with streptavidin horseradish peroxidase (VectorLabs, 1:300) and DAB chromogen with nickel (Vector Labs). Sections werethen coverslipped and examined under a light microscope. Followingtissue punching the fresh-frozen blocks were sectioned at 20 um.Sections were stained with H&E and for DDC-IR.

[0187] Quantitative estimates of the total number of AAV-infected cellswithin the caudate nucleus, putamen and globus pallidus were determinedby using an optical dissector procedure. The optical dissector systemconsisted of a computer assisted image analysis system, including anLeitz Otholux 11 microscope hard-coupled to a Prior H128computer-controlled x-y-z motorized stage, a high sensitivity Sony 3CCDvideo camera system (Sony, Japan) and a Macintosh G-3 computer. Allanalyses were performed using NeuroZoom software (La Jolla, Calif.).Prior to each series of measurements, the instrument was calibrated. Theregion of positive neurons in the caudate, putamen and globus palliduswas outlined at low magnification (2.5× objective). Because of thediffuse presence of AAV-infected cells within the striatum, 1% of theoutlined region was measured with a systematic random design ofdissector counting frames (1 505 1IM2) using a 63× plan-neofluarimmersion objective with a 0.95 numerical aperture. Based on pilotexperiments at least four sections equally spaced were sampled. By usingthe dissector principle, up to 200 AADC positive neurons were sampled byoptical scanning by using uniform, systematic and random designprocedures for all measurements. The average thickness of the sectionswas measured at 23 microns. Once the top of the section was in focus,the z-plane was lowered a 1-2 gm. Counts were than made while focusingdown through three 5 lim-thick dissectors. Care was taken to ensure thatthe bottom forbidden plane was never included in the analysis. Thevolumes of the structures were calculated according to standardprocedures. The total number of positive cells in the examinedstructures was calculated by using the formula N=Nv×Vs, where Nv is thenumerical density and Vs is the volume of the structure.

[0188] TH-IR staining revealed robust reduction of the nigrostriatalfibers and cell bodies in the substantia nigra on the ipsilateral sidein all of the monkeys. The contralateral side showed variable reductionsof TH and AADC-IR in the striatum and the substantia nigra.

[0189] DDC-IR paralleled TH-IR only in the monkeys treated withAAV-LacZ. The AAV-AADC-treated monkeys showed robust AADC staining onthe ipsilateral side that exceeded staining seen on the contralateralside. A high density of AADC-IR cells was seen throughout 80% of thestriatum and 100% of the globus pallidus in one of the AAV-AADC treatedanimals. Stereological analysis revealed 18,384 cells per mm³ in theputamen, 15,126 cells per mm³ in the caudate and 9,511 cells per mm³ inthe globus paillidus. The total number of AAV-infected cells wasestimted to be at least 16×10⁶ cells. In the other AAV-AADC treatedmonkey, AADC-IR cells were found in over 60% of the ipsilateralstriatum, with 7,515 cells per mm³ in the caudate and 15,352 cells permm in the putamen and 3,850 cells per mm³ in the globus pallidus. NoAADC cells were found in the contralateral striatum. TheAAV/LacZ-treated monkeys did not show AADC-IR in either the ipsilateralor contralateral striata.

[0190] Cells infected with AAV appeared to have neuronal morphology. Theaverage diameter of the infected cells was 9±2.3 μm in the putamen and14.6±9 μm. Many Lac-Z and AADC cells had a typical medium spiny neuronmorphology. AAV-infected cells were positive for the neuronal marker,Neu-N. In the AAV-AADC-treated monkeys, one out of 4-6Neu-N-positivecells was AADC-positive in the caudate and putamen, and one out of 3-4Neu-N-positive cells was AADC positive in the globus pallidus. None ofthe AAV-infected cells in the Lac-Z or AADC-treated monkeys wereGFAP-positive.

[0191] Areas adjacent to cannula tracts were stained with Nissi and H&Estaining. No signs of cytotoxicity were observed. No perivascularcuffing was observed, regardless of the distance from the cannula. Therewere no signs of neuronal cell reduction close to the infusion site whencompared to the contralateral side using Neu-N immunostaining.GFAP-immunostaining failed to detect any abnormal glia reaction withinthe AAV-treated striatum.

Biochemical Analysis

[0192] Brain regions were removed from fresh frozen blocks using amicropuncher in order to evaluate tissue levels of L-dopa and dopaminemetabolites and the activity of AADC and the presence of the AAV-vector.Brain regions included striatum and cortex.

[0193] Frozen micropunches were collected, and homogenized by ultrasonicprocessing in 300 pl of 0.1 M perchloric acid (Fisher Scientific)containing 1% ethanol, and 0.02% EDTA (Fisher Scientific). Fifty pl ofthe homogenate was removed for protein analysis (BCA Protein Assay KitPierce #23225), and the remainder centrifuged in a mirocentrifuge for1.5 minutes at maximum speed. 30 to 50 pl of the homogenate was used forcatecholamine analysis by HPLC using an Ultrasphere C-18 ion pair, 5 p,4.6×250 mm column (Beckman 235329); a Waters 717 plus autosampler at 4°C., Waters 510 pump at 0.9 mv, min, and amperometric electrochemicaldetector (Decade) set at Eox. 0.82V. The column and detector cell wereset at 31° C. The mobile phase contained 2 L HPLC grade water, 2.2 g 1-heptanesulfonic acid, sodium salt (Fisher Scientific), 0.17 g EDTA, 12ml triethylamine (Fisher Scientific), pH adjusted to 2.5 with═−8 ml 85%phosphoric acid (Fisher Scientific), and 60 ml acetonirile (J. T.Baker). The detector output was recorded and analyzed with the WatersMillennium 32 Chromatography Manager.

[0194] AADC analysis

[0195] AADC activity was determined by an adaptation of the method ofNagatsu et al (1979) Anal. Biochem. 100:160-165. Briefly, tissue (10mg/ml) was homogenized in 50 mM phosphate buffer (pH 7.4) containing0.04 mM pyrixyl phosphate (a AADC cofactor) and 0.2 mM pargyline.Samples were pre-incubated at 37° C. for 5 minutes and the reaction wasinitiated by the addition of L-dopa (final concentration; 100μM).Incubations were carried out for 20 minutes and the reaction stopped bythe addition of 0.02 ml concentrated perchloric acid. Aftercentrifugation, the supernatant dopamine concentration was determinedusing HPLC with electrochemical detection. (see, e.g., Boomsa et al.(1988) Clin. Chem. Acta 178-59-69). Protein concentration in the tissuepellet was determined using the BCA Protein Assay Kit (Pierce #23225).Results are expressed as nM/hr/mg of protein. Frozen tissue punches wereprocessed according to standard protocols.

[0196] Cortical regions of all monkeys showed variable levels of L-dopa,however, they were consistent within each monkey. As expected, there wasno decarboxylation of L-dopa to dopamine within the cortex, however, inthe striatum on the side contralateral to MPTP administration, L-dopawas converted to dopamine and further metabolized to HVA. In theMPTP-treated striatum of the AAV-Lac-Z monkeys, L-dopa was not convertedto dopamine, nor was it metabolized to HVA. Tissue levels of L-dopa alsoremained at the same levels as in the cortex in AAV-Lac-Z treatedmonkeys. In the MPTP-treated striatum of AAV-AADC-treated monkeys,L-dopa was converted to dopamine and HVA and tissue levels of L-dopa inthis region were reduced.

[0197] AADC activity was very low in the cortical regions and in theMPTP-treated striatum of AAV-LacZ-treated monkeys. L-dopa was convertedto dopamine in the contralateral striatum, suggesting high levels ofAADC activity. The tissue punches from MPTP-treated striatum of AAV-AADCinfected monkeys contained extremely high dopamine levels with onlytraces of L-dopa left.

[0198] These results demonstrate that the combination of infusedAAV-AADC vector and systemic L-dopa is a promising therapy for thetreatment of PD.

[0199] Thus, the invention provides a novel and efficient treatmentmethod for CNS disorders, such as Parkinson's Disease. In addition, theinvention also provides methods for determining dopamine activity invivo.

1 12 1 31 DNA Artificial Sequence Description of ArtificialSequenceprimer/probe 145A 1 aagtcatcgg ctcgggtacg tagacgatat c 31 2 30DNA Artificial Sequence Description of Artificial Sequenceprimer/probetk 2 atagcagcta caatccagct accattctgc 30 3 30 DNA Artificial SequenceDescription of Artificial Sequenceprimer/probe 145A 3 gctcggtacccgggcggagg ggtggagtcg 30 4 30 DNA Artificial Sequence Description ofArtificial Sequenceprimer/probe 145B 4 taatcattaa ctacagcccg gggatcctct30 5 24 DNA Artificial Sequence Description of ArtificialSequenceprimer/probe P547 5 ggtttgaacg agcgctcgcc atgc 24 6 42 DNAArtificial Sequence Description of Artificial Sequenceprimer/probe blunt1 6 cgcgccgata tcgttaacgc ccgggcgttt aaacagcgct gg 42 7 42 DNAArtificial Sequence Description of Artificial Sequenceprimer/probe blunt2 7 cgcgccagcg ctgtttaaac gcccgggcgt taacgatatc gg 42 8 37 DNAArtificial Sequence Description of Artificial Sequenceprimer/probe5DIVE2 8 tgtggtcacg ctgggggggg gggcccgagt gagcacg 37 9 31 DNA ArtificialSequence Description of Artificial Sequenceprimer/probe polylinker 1 9ccgctacagg gcgcgatatc agctcactca a 31 10 58 DNA Artificial SequenceDescription of Artificial Sequenceprimer/probe polylinker 2 10ggatccggta ccgcccgggc tctagaatcg atgtatacgt cgacgtttaa accatatg 58 11 34DNA Artificial Sequence Description of Artificial Sequenceprimer/probeE4.1 11 agaggcccgg gcgttttagg gcggagtaac ttgc 34 12 22 DNA ArtificialSequence Description of Artificial Sequenceprimer/probe E4.2 12acatacccgc aggcgtagag ac 22

What is claimed is:
 1. A method for delivering recombinant AAV virionsto a subject, comprising administering via convection-enhanced delivery(CED) said rAAV virions into the CNS of the subject, wherein said rAAVvirions comprise a nucleic acid sequence encoding a therapeuticpolypeptide.
 2. The method of claim 1, wherein the administering is donewith an osmotic pump.
 3. The method of claim 1, wherein theadministering is done with an infusion pump.
 4. The method of claim 1,wherein the nucleic acid sequence encodes an aromatic-amino-aciddecarboxylase (AADC).
 5. The method of claim 1 wherein the subject is ahuman.
 6. The method of claim 1, wherein the rAAV virions areadministered into the striatum.
 7. A method for delivering recombinantAAV virions to a subject having a CNS disorder, comprising administeringvia convection-enhanced delivery (CED) said virions into the CNS of thesubject, wherein said virions comprise a nucleic acid sequence encodinga therapeutic polypeptide.
 8. The method of claim 7 wherein the CNSdisorder is Parkinson's disease, the rAAV virions are administered intothe striatum and wherein the nucleic acid sequence encodes AADC
 9. Amethod for treating a neurodegenerative disease in a subject, saidmethod comprising: (a) providing a preparation comprising recombinantadeno-associated virus (rAAV) virions, wherein said virions comprise anucleic acid sequence that is expressible in transduced cells to providea therapeutic effect in the subject; and (b) delivering the preparationto the CNS of the subject using convection-enhanced delivery (CED),wherein said virions transduce neural cells and the nucleic acidsequence is expressed to provide a therapeutic effect in the subjectsuitable for treating said neurodegenerative disease.
 10. The method ofclaim 9, wherein the neurodegenerative disease is Parkinson's disease.11. The method of claim 9, wherein the nucleic acid sequence expressiblein transduced cells encodes AADC or functional fragment thereof.
 12. Themethod of any one of claims 9-11, further comprising administering tothe subject at least one additional therapeutic compound.
 13. The methodof claim 12 wherein the at least one additional therapeutic compound isL-dopa.
 14. The method of claim 13, further comprising administeringL-dopa and, optionally, carbidopa to the subject.
 15. A method ofdetermining levels of dopamine activity in the brain of subjectcomprising; (a) administering a labeled tracer to the subject, whereinbinding of the tracer to a cell is indicative of dopamine activity; and(b) imaging the subject's brain to determine the number of cells whichbind the labeled tracer, thereby determining levels of dopamine activityin the subject's brain.
 16. The method of claim 15, wherein the labeledtracer is 6-[¹⁸F]-fluoro-L-m-tryosine (¹⁸F-FMT).
 17. The method of claim15, wherein the imaging is positron emission tomograph (PET) imaging.